Differentiation of human ips cells to human alveolar type ii via definitive endoderm

ABSTRACT

The present invention relates to compositions and methods for generating populations of tissue precursor cells from pluripotent cells, and preferably induction of stem cells into definitive endoderm to generate anterior foregut endoderm from pluripotent cells. The anterior foregut endoderm cells can then be differentiated into an alveolar epithelial type II cell.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of International PatentApplication No. PCT/US2013/061687, filed on Sep. 25, 2013, which isentitled to priority under 35 U.S.C. § 119(e) to U.S. ProvisionalApplication Ser. No. 61/705,427, filed Sep. 25, 2012, the each of whichis hereby incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under EB008366,HL107205, HL107768, HL111016, HL083895, and HL098220 awarded by NationalInstitutes of Health. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Lung disease is the third-leading cause of death in the United States,with more than 400,000 deaths annually (Longmire T A, et al. 2012. CellStem Cell 10(4):398-411, Petersen T H, et al. 2011. Material today14(5):196-201). Lung transplantation is a possible treatment for peoplewho have end-stage lung disease. Lung transplantation is limited by thelow availability of donor lungs. Moreover, surgical, medical andimmunological complications cause considerable morbidity and mortalityin this population. As a result, many patients die each year while on awaiting list or because of transplant complications (Longmire T A, etal. 2012. Cell Stem Cell 10(4):398-411, Nichols J E, et al. 2012. J CellBiochem 113(7):2185-2192, McCurry K R, et al. 2009. Am J Transplant9(Part 2):942-958).

Transplantation of adult lung stem and progenitor cells or alveolarcells, isolated from human lung, is emerging as an alternative to wholeorgan transplantation (Wang D, et al. 2007. Proc Natl Acad Sci USA.104(11):4449-4454). However, this approach is also limited by thescarcity of human epithelial cells, and the difficulties of expandingthese cells in vitro. Moreover, the successful engraftment of such cellsin vivo in injured lungs has not yet been demonstrated (Wang D, et al.2007. Proc Natl Acad Sci USA. 104(11):4449-4454, Tesei A, et al. 2009.Cell Prolif 42(3):298-308, Fujino N, et al. 2012. Am J Respir Cell MolBiol 46(4):422-430).

One potential future treatment for severe lung disease istransplantation with engineered lungs that are capable of gas exchange.To avoid immunological rejection, such engineered lungs should becreated using individual-specific (autologous) lung and airway cells(Nichols J E, et al. 2012. J Cell Biochem 113(7):2185-2192, Petersen TH, et al. 2010. Science 329(5991):538-541, Badylak S F, et al. 2012.Lancet 379(9819):943-952). Therefore, a significant emphasis is beingplaced on identifying a reliable source of functional lung epithelialcells to be used in lung-related therapies (Petersen T H, et al. 2011.Material today 14(5):196-201, Kotton D N, et al. 2012. Am J Respir CritCare Med 185(12):1255-1260).

Induced pluripotent stem (iPS) cells are the product of adult somaticcell reprogramming to an embryonic-like state by inducing a “forced”expression of specific pluripotent genes (Takahashi K, et al. 2007. Cell131(5):861-872, Yu J, et al. 2007. Science 318(5858):1917-1920). It ispostulated that the use of human iPS cells may be the most effectivestrategy to develop respiratory epithelial cells that may be valuable inlung-related cell therapies and tissue engineering (Nishikawa S, et al.2008. Nat Rev Mol Cell Biol 9(9):725-729, Green M D, et al. 2011. NatBiotechnol 29(3):267-272, Mou H, et al. 2012. Cell Stem Cell10(4):385-397). Given that iPS cells can be derived from the patient tobe treated, they could provide a cell source that is geneticallyidentical to the patient, allowing tissue generated from these cells toavoid immune rejection (Badylak S F, et al. 2012. Lancet379(9819):943-952, Yu J, et al. 2007. Science 318(5858):1917-1920).

The differentiation of human embryonic stem and iPS cells (hESCs andiPSCs, respectively) into pulmonary epithelium has been challenging.Several research groups have reported the successful differentiationtoward a range of pulmonary epithelial cell types, including bothalveolar type II cells (AETII cells) and other airway epithelium, usinga variety of protocols (Longmire T A, et al. 2012. Cell Stem Cell10(4):398-411, Wang D, et al. 2007. Proc Natl Acad Sci USA.104(11):4449-4454, Green M D, et al. 2011. Nat Biotechnol 29(3):267-272,Mou H, et al. 2012. Cell Stem Cell 10(4):385-397, Van Haute L, et al.2009. Respir Res 10:105, Ali N N, et al. 2002. Tissue Eng 8(4):541-550,Rippon H J, et al. 2006. Stem Cells 24(5):1389-1398, SamadikuchaksaraeiA, et al. 2006. Tissue Eng 12(4):867-875). However, conditions fordirecting hESCs or iPSCs to differentiate along an alveolar epitheliallineage with high homogeneity have not yet been reported, and mostprotocols generate a mixed population of epithelial cells from hESCs oriPSCs.

Recently, the focus in organ engineering has centered on decellularizingcomplex organs such as heart, liver, and kidney, and using the acellularmatrices as scaffolds for repopulation with organ-specific cells.Because the decellularized organ has the extracellular matrix template,it contains appropriate three-dimensional (3D) architecture andregionally-specific sites for cellular adhesion (Nichols J E, et al.2012. J Cell Biochem 113(7):2185-2192, Petersen T H, et al. 2010.Science 329(5991):538-541). With extracellular matrix derived from donorlungs, the capacity to regenerate lung tissue from autologous cells(e.g., autologous iPS-derived epithelium) would therefore constitute amajor medical advance. One way to accomplish this in lung engineering isto differentiate human iPSCs into respiratory epithelial cells and/orinto putative postnatal stem cells of the respiratory system, and toreseed the lung acellular matrix with these cells (Badylak S F, et al.2012. Lancet 379(9819):943-952).

There is a need in the art for regeneration of lung tissue fromautologous cells. The present invention addresses this unmet need in theart.

SUMMARY OF THE INVENTION

The invention provides a method of differentiating a population of stemcells into a population of lung cells, the method comprising: a)inducing a stem cell into a definitive endoderm cell; b) inducing thedefinitive endoderm cell into an anterior foregut endoderm cell; c)inducing the anterior foregut endoderm cell into a lung cell, therebydifferentiating a stem cell into a lung cell.

In one embodiment, the stem cell is cultured without serum in thepresence of Activin A in order to induce the stem cell into a thedefinitive endoderm cell.

In one embodiment, the definitive endoderm cell is cultured in thepresence of an extracellular matrix (ECM) protein and a culture mediumsupplemented with an inhibitor of bone morphogenic protein (BMP) and aninhibitor of TGF-β signaling in order to induce the definitive endodermcell into an anterior foregut endoderm cell.

In one embodiment, the inhibitor of BMP is NOGGIN and the inhibitor ofTGF-β signaling is SB-431542.

In one embodiment, the ECM protein is human ECM selected from the groupconsisting of collagen, laminin, fibronectin, tenascin, elastin,proteoglycan, glycosaminoglycan, and any combination thereof.

In one embodiment, the anterior foregut endoderm cell is cultured in thepresence of a differentiation medium comprising FGF-10, EGF, Wnt3a, andKGF in order to induce the anterior foregut endoderm cell into a lungcell wherein the lung cell is an alveolar epithelial type II cell.

In one embodiment, the differentiation medium does not include BMP4.

In one embodiment, the alveolar epithelial type II cell is an alveolarepithelial type II progenitor cell.

In one embodiment, the population of lung cells is at least 95% of cellsexhibiting an alveolar type II phenotype.

In one embodiment, the alveolar type II phenotype is expression of analveolar type II cell marker selected from the group consisting of SPC,Mucin-1, SPB, CD54, and any combination thereof.

In one embodiment, the lung cell is cultured on a decellularized lungmatrix.

The invention provides a population of lung cells produced by a methodof differentiating a stem cell into a lung cell, the method comprising:a) inducing a stem cell into a definitive endoderm cell; b) inducing thedefinitive endoderm cell into an anterior foregut endoderm cell; c)inducing the anterior foregut endoderm cell into a lung cell, therebydifferentiating a stem cell into a lung cell.

In one embodiment, the population is at least 95% of cells exhibiting analveolar type II phenotype.

In one embodiment, the alveolar type II phenotype is expression of analveolar type II cell marker selected from the group consisting of SPC,Mucin-1, SPB, CD54, and any combination thereof.

In one embodiment, the population of cells comprises geneticallymodified cells.

In one embodiment, the cells are genetically modified to express atherapeutic gene.

In one embodiment, the cells resemble freshly isolated human primaryalveolar type II cells.

The invention also provides a method of alleviating or treating a lungdefect in a mammal, the method comprising administering to the mammal atherapeutically effective amount of a composition comprising apopulation of lung cells produced by a method of differentiating a stemcell into a lung cell, thereby alleviating or treating said lung defectin said mammal, wherein the differentiation method comprise: a) inducinga stem cell into a definitive endoderm cell; b) inducing the definitiveendoderm cell into an anterior foregut endoderm cell; c) inducing theanterior foregut endoderm cell into a lung cell, thereby differentiatinga stem cell into a lung cell.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1, comprising FIGS. 1A through 1D, is a series of images depictingschematics summarizing the experiments. FIG. 1A is an image depicting aschematic protocol for directed differentiation of iPSCs to AETII invitro in 22 days. Cytokines were added at different steps indicated ontop of panel. FIG. 1B is an image depicting the summary of lungdevelopmental steps and corresponding markers at each step. FIG. 1C isan image depicting a schematic summarizing the iPSC differentiation anddecellularization-recellularization of both rat and human lung withiPSC-derived AETII cells. FIG. 1D is a series of phase-contrast imagesof iPSCs at day 0, DE cells at day 6, and differentiated cells at day 15and 22, which are termed AETII cells. Scale bar, 63 μm.

FIG. 2, comprising FIGS. 2A through 2O, is a series of images depictingthe characterization of cells at day 8 of differentiation to produceanterior foregut endoderm (AFE). (FIGS. 2A-2I) Immunofluorescenceanalysis of AFE markers; (FIGS. 2A, 2D, 2G) show DAPI staining fornuclei; (FIGS. 2B, 2E, 2H) show PAX9, TBX1 and SOX2 positive cells;(FIGS. 2C, 2F, 2I) merge at day 8. (FIG. 2J) Immunofluorescence stainingshowing AFE cells are positive for both SOX2 and FOXA2. (FIG. 2K) Flowcytometric analysis of double positive cells for SOX2 and FOXA2 in AFEcells at day 8 compared to cells cultured in activin A and RPMI mediumonly (Y axis: % positive cells for FOXA2/SOX2). (FIG. 2L) mRNAexpression of SOX2, TBX1 and PAX9 in AFE generated from DE cells invitro at day 8 (data expressed as quantification of mRNA normalized toGAPDH and average fold change in gene expression over iPS cells. Y axis:fold changes in gene expression compared with iPSC) (FIG. 2M) Expressionof NKX2.1 on day 13 after induction anterior foregut endoderm quantifiedby DAPI staining for nuclei, NKX2.1 positive cells, and merge. (FIG. 2N)Immunofluorescence staining showing NKX2.1 cells in AFE stained positivefor FOXA2 indicating these cells are more lung progenitor rather thanthyroid progenitor. (FIG. 2O) Flow cytometric analysis of positive cellsfor NKX2.1. Up to 24% of AFE cells were positive for NKX2.1 at day 13.(Y axis: percentages of positive cells for NKX2.1). Bars indicatemean±SEM of n=3 independent experiments for qRT-PCR and flowcytometry. * denotes statistically significant difference p-value <0.05.Scale bar, 31 μm.

FIG. 3, comprising FIGS. 3A through 3K, is a series of imaged depictingfunctional characterization of AETII cells derived from iPSCs, day 22 ofdifferentiation (C1 clone). (FIGS. 3A-3D) Immunostaining of alveolartype II marker, (FIG. 3A) ProSPC, (FIG. 3B) Mucin-1, (FIG. 3C) proSPA,(FIG. 3D) ProSPB. (Scale bar, 63 μm) (FIGS. 3E-3F) Transmission electronmicroscopy represent (FIG. 3E) human AETII and (FIG. 3F) iPSC-derivedAETII containing characteristic cytoplasmic laminar bodies (Scale bar,0.5 μm). (FIG. 3G) qRT-PCR analysis in undifferentiated iPSC, DE, AFEand differentiated AETII cells compared to human AETII, from threeindependent experiments. Values from the triplicate PCR reactions for agene of interest (SPA, SPB, SPC, and Mucin-1) were normalized againstaverage GAPDH Ct values from the same cDNA sample. Fold change of GOItranscript levels between iPS derived-AETII and human type II cellsequals 2^(−ΔΔCt), where ΔCt=Ct_((GOI))−Ct_((GAPDH)), andΔΔCt=ΔCt_((AETII))−ΔCt_((ATII)) (FIG. 3H) Flow cytometry analysis forthe percentage of positive cells for alveolar type II and type I markersat day 22. Cells were negative for, p63 and SOX2. (FIG. 3I) Expressionof albumin, CD31, TSHR, and CC10 (CCSP) in iPSC-AETII. Cells werenegative for genes indicative of other lineages at day 22. (FIG. 3J)Amount of secreted SPC in the iPSC-derived AETII supernatants collectedduring the time course of differentiation compared to human type IIcells determined by ELISA. (FIG. 3K) Western blot for proSPC iniPSC-AETII at day 22 and β-actin as an internal control. Bars indicate±SEM and n=3 independent experiments for qRT-PCR, ELISA and flowcytometry. * denotes statistically significant difference p-value <0.05.

FIG. 4, comprising FIG. 4A through FIG. 4D, is a series of imagesdepicting the functional characterization of AETI cells derived fromiPSCs, day 29 of differentiation (C1 clone). (FIG. 4A-4B)Immunofluorescent staining of alveolar type I marker (FIG. 4A) T1α (FIG.4B) Caveolin-1 (Scale bar, 63 μm). (FIG. 4C) Flow cytometry analysis forthe percentage of positive cells for alveolar type I marker at day 29 inthe presence and absence of IWR-1 (Y axis: % of positive cells). (FIG.4D) qRT-PCR analysis in AETI cells as compared to native human type I(AETI) cells, from three independent experiments. Values from thetriplicate PCR reactions for a gene of interest (AQ5, T1α, Caveolin-1)were normalized against average GAPDH Ct values from the same cDNAsample. Fold change of GOI transcript levels between iPS derived-AETIand human type I cells equals 2^(−ΔΔCt), whereΔCt=Ct_((GOI))−Ct_((GAPDH)), and ΔΔCt=ΔCt_((AETI))−ΔCt_((hAETI)) (Yaxis: relative gene expression compared with human type I cells). Barsindicate ±SEM and n=3 independent experiments for qRT-PCR, ELISA andflow cytometry.

FIG. 5, comprising FIG. 5A through FIG. 5P, is a series of imagesdepicting iPSC-derived AETII recellularized 3D rat lung tissue scaffoldsin a bioreactor (FIG. 5A) H&E staining of decellularized rat lung; (FIG.5B-5C) H&E staining of 3- and 7-day seeded rat lung with iPSC-derivedAETII cells cultured in a bioreactor (scale bar 25 μm). (FIG. 5D-5F).Immunofluorescent staining for pro-SPC in AETII seeded cells at day 3(FIG. 5D) DAPI staining; (FIG. 5E) Pro-SPC; (FIG. 5F) merge (arrows inFIG. 5F indicate positive cells for pro-SPC). (FIG. 5G-I) Immunostainingfor NKX2.1 at day 7. (FIG. 5G) DAPI, (FIG. 5H) NKX2.1, (FIG. 5I) merge(arrows in FIG. 5I indicate positive cells for NKX2.1) (FIG. 5J-5M)Caspase and PCNA immunostaining at day 7, (arrows indicates positivecells for PCNA in FIG. 5L and caspase in FIG. 5M) (Scale bar, 25 μm).(FIG. 5N) Proliferation at day 7 compared with day 3. iPSC-AETIIdisplayed a significantly increased fractional proliferation (P<0.05)after 7 days when they were stained for PCNA (Y axis: % proliferationbased on the number of positive nuclei stained for PCNA). (FIG. 5O)Immunostaining of the few engrafted epithelial cells that acquiredflattened morphology, positive for T1α and negative for NKX2.1 at day 7(Scale bar, 63 μm, arrows in FIG. 5O indicate positive cells for T1α).(FIG. 5P) Flow cytometry for SPC, T1α, CCSP, p63 and SOX2 before andafter seeding into rat lung scaffold in bioreactor. The number of SPCpositive cells decreased during 7-day culture, while the number ofpositive cells for T1α increased from 9% to 31.2%. All differentiatedcells from iPS cells were negative for CCSP, p63 and SOX2 before andafter cell seeding.

FIG. 6, comprising FIG. 6A through FIG. 6V, is a series of imagesdemonstrating that iPSC-derived AETII (C1 clone) adhere to sections ofacellular rat and human lung matrix. (FIG. 6A-G) iPSC-AETII on humanlung sections at day 7. (FIG. 6A) H&E, scale bar 200 μm (FIG. 6B)Immunostaining for SPC and CCSP. (FIG. 6C) Immunostaining for NKX2.1 andT1a (arrows indicates positive cells for SPC in FIG. 6B and T1a in FIG.6C, scale bar, 63 μm). (FIG. 6D-6F) Immunostaining for NKX2.1. (FIG. 6D)DAPI, (FIG. 6E) NKX2.1, (FIG. 6F) merge, scale bar 50 μm (FIG. 6G)Caspase and PCNA immunostaining, scale bar 49 μm. (FIG. 6H-6O)iPSC-derived AETII cultured on rat lung sections for 7 days. (FIG. 6H)H&E, scale bar 200 μm (FIG. 6I) Immunostaining for SPC and CCSP. (FIG.6J-6K) Immunostaining for NKX2.1 and T1a (arrows indicates positivecells for SPC in FIG. 6I, T1α in FIG. 6J and NKX2.1 in FIG. 6K), scalebar 63 μm. (FIG. 6L) DAPI staining (FIG. 6M) Immunostaining for PCNA and(FIG. 6N) caspase, (FIG. 6O) merge (Scale bar 50 μm). (FIG. 6P-6V)Native human AETII cells, isolated from fresh adult human lung, culturedon human lung sections for 7 days. (FIG. 6P) H&E, scale bar 200 μm.(FIG. 6Q) Immunostaining for SPC and CCSP, scale bar 63 μm (FIG. 6R)Immunostaining for NKX2.1 and T1α, scale bar 63 μm (arrows indicatepositive cells for SPC in FIG. 6Q and T1α in FIG. 6R). (FIG. 6S-6U)Immunostaining for NKX2.1. (FIG. 6S) DAPI, (FIG. 6T) NKX2.1, (FIG. 6U)merge, scale bar 50 μm. (FIG. 6V) Caspase and PCNA immunostaining, scalebar 49 μm.

FIG. 7, comprising FIG. 7A through FIG. 7M, is a series of imagesdepicting the functional characteristics of definitive endoderm (DE)cells derived from iPSCs (C1 clone), at day 6. (FIG. 7A-7F)Immunofluorescence analysis of DE marker proteins, SOX17 and FOXA2 atday 6. (FIG. 7A and FIG. 7D) Nuclei were stained with DAPI, (FIG. 7B andFIG. 7E) shows SOX17, FOXA2 staining in DE cells, (FIG. 7C and FIG. 7E)Merge. (FIG. 7G-7J) immunofluorescence staining showing DE cells arepositive for both SOX17 and FOXA2 at day 6, (FIG. 7K) flow cytometricanalysis of double positive cells for SOX17/FOXA2 in DE cells exposed toactivin A at day 6 compare to iPS cultured in media without activin A,(FIG. 7L) mRNA expression of SOX17, FOXA2 and CXCR4 from threeindependent experiments by qRT-PCR. (Data expressed as quantification ofmRNA normalized to GAPDH and average fold change in gene expression overiPSCs), (FIG. 7M) Flow cytometric analysis of SOX17, FOXA2 and CXCR4during activin A-mediated induction of definitive endoderm in iPSC cellsat day 6 (compare to DE cells stained with corresponding isotype). Barindicate ±SEM and n=3 independent experiments for qRT-PCR and flowcytometry. * on the graph denotes statistically significant differencep-value <0.005, Scale bar, 31 μm.

FIG. 8, comprising FIG. 8A through FIG. 8M, is a series of imagesdepicting the characteristics of definitive endoderm cells derived fromiPSCs C2 clone, at day 6: (FIG. 8A-8F) Immunofluorescent staining ofdefinitive endodermal markers, SOX17 and FOXA2 at day 6 of activin Ainduction (Scale bar, 31 μm), (FIG. 8G-8J) Immunofluorescence stainingshowing DE cells are positive for both SOX17 and FOXA2, (FIG. 8K) flowcytometric analysis of double positive cells for SOX17/FOXA2 in DE cellsexposed to activin A at day 6 compare to iPS cultured in media withoutactivin A, (FIG. 8L) Expression of SOX17, CXCR4 and FOXA2 mRNA in C2iPSCs quantified by qRT-PCR at day 6. (Data expressed as quantificationof mRNA normalized to GAPDH and average fold change in gene expressionover iPS cells), (FIG. 8M) Representative flow cytometric analysis ofSOX17, CXCR4 and FOXA2 in C2 iPSCs derived DE at 6 day. Bar indicate±SEM and n=3 independent experiments for qRT-PCR and flow cytometry. *on the graph denotes statistically significant difference p-value<0.005, Scale bar, 31 μm.

FIG. 9, comprising FIG. 9A through FIG. 9O, is a series of imagesdepicting the analysis of AFE markers in NOGGIN/SB-431542-treateddefinitive endoderm in C2 iPS cells. (FIG. 9A-9I) Immunofluorescentstaining of AFE markers; SOX2, TBX1, PAX9, after 2 day ofNOGGIN/SB431542 induction in C2 iPS cells (at day 8). Scale bar, 31 μm,(FIG. 9J) Immunofluorescence staining showing AFE cells are positive forboth SOX2 and FOXA2 at day 8, (FIG. 9K) Flow cytometric analysis ofdouble positive cells for SOX2/FOXA2 in AFE cells at day 8. More than85% of cells were double positive for both SOX2 and FOXA2. (FIG. 9L)Expression of TBX1, SOX2, and PAX9 mRNA quantified by qRT-PCR in C2 iPScells at day 8 (Data expressed as quantification of mRNA normalized toGAPDH and average fold change in gene expression over iPSCs cells).(FIG. 9M) Immunofluorescence staining of NKX2.1 in AFE derived fromclone C2 at day 13 (Scale bar, 31 μm). (FIG. 9N) Immunofluorescencestaining showing AFE cells are positive for both NKX2.1 and FOXA2 at day13; Most NKX2.1 positive cells were stained positive for FOXA2 (Scalebar, 31 μm). (FIG. 9O) Flow cytometric analysis of positive cells forNKX2.1 in AFE cells at day 13. Exposing DE to NOGGIN/SB431542 yield 26%positive cells for NKX2. Bar indicate ±SEM and n=3 biological triplicatereplicates for qRT-PCR and flow cytometry, * denotes statisticallysignificant difference p-value <0.05.

FIG. 10, comprising FIG. 10A through FIG. 10C, is a series of graphsdemonstrating the differentiation of DE cells (day 6) to AETII (day 22)on different extracellular matrix proteins. (FIG. 10A) SPC, (FIG. 10B)SPB and (FIG. 10C) NKX2.1 expression in AETII differentiated on collagenI, collagen IV, fibronection, and human ECM protein and matrigel,quantified qRT-PCR. The iPSC-derived DE differentiated to AETII ondifferent ECM protein. The gene expression in iPS derived-AETII cells ondifferent ECM proteins were compared to the level seen in iPSderived-hAETII cells on matrigel. Ct values from three independentexperiments from the triplicate PCR reactions for a gene of interest(SPB, SPC, and NKX2.1) were normalized against average GAPDH Ct valuesfrom the same cDNA sample. Fold change of GOI transcript levels betweeniPS derived-AETII on each ECM protein and iPS derived-AETII cells onmatrigel equals 2^(−ΔΔCt), where ΔCt=Ct_((GOI))−Ct_((GAPDH)), andΔΔCt=ΔCt_((iPSC-AETII on ECM of interest))−ΔCt_((iPSC-AETII on Matrigel)).Human ECM induced significantly higher levels of SPC, SPB and NKX2.1expression compared to each ECM proteins individually. (Bar indicate±SEM and n=3 independent experiments).

FIG. 11, comprising FIG. 11A through FIG. 11J, is a series of imagesdepicting the functional characterization of differentiated AETII fromC2 iPSCs line. (FIG. 11A) Phase-contrast images AETII cells. (FIG.11B-11C and FIG. 11E-11F) Immunofluorescent staining of alveolar type IImarkers; (FIG. 11B) Pro surfactant protein B (ProSPB), (FIG. 11C)Mucin-1, (FIG. 11E) Surfactant protein A (SPA), (FIG. 11F) Prosurfactant protein C (ProSPC) Scale bar, 63 μm, (FIG. 11D) Transmissionelectron microscopy, represent AETII contain characteristic cytoplasmiclaminar bodies (scale bar, 1 μm) (FIG. 11G) qRT-PCR analysis inundifferentiated iPSC, DE, AFE and AETII cells derived from C2 clonecompared to hATII cells that were derived from fresh human lung, fromthree independent experiments values from the triplicate PCR reactionsfor a gene of interest (SPA, SPB, SPC, Mucin-1) were normalized againstaverage GAPDH Ct values from the same cDNA sample. Fold change of GOItranscript levels between iPS derived-AETII and human type II cellsequals 2^(−ΔΔCt), where ΔCt=Ct_((GOI))−Ct_((GAPDH)), andΔΔCt=ΔCt_((AETII))−ΔCt_((ATII)), (FIG. 11H) Flow cytometry analysis forthe percentage of positive cells for alveolar type II markers at day 22.More than 95% of population were positive for type II cells marker(CD54, SPB, SPC, Mucin-1) when they were negative for CCSP (Clara cellmarker), p63 (basal stem cell marker), (FIG. 11I) Expression of albumin,CD31, TSHR, CC10 (CCSP) in iPSC-derived AETII; they were negative forgenes indicative of other lineages at day 22. (FIG. 11J) The amount ofsecreted SPC in the iPSC-derived AETII during the time course ofdifferentiation compared to SPC secretion from isolated AETII from humanlung determined by enzyme-linked immunosorbent assay. Bars indicate ±SEMand n=3 independent experiments for qRT-PCR, ELISA and flow cytometry. *denotes statistically significant difference p-value <0.05.

FIG. 12, comprising FIG. 12A through FIG. 12D, is a series of graphsdepicting the results of experiments. (FIG. 12A-12B) Sequential up anddownregulation of DE-specific and AFE-specific genes duringdifferentiation to AETII cells quantified by qRT-PCR. (FIG. 12A-12B)Ratio of gene expression in DE cells compare to iPSCs duringdifferentiation quantified by qRT-PCR in (FIG. 12A) C1 clone and (FIG.12B) C2 clone (Data expressed as quantification of mRNA normalized toGAPDH and average fold change in gene expression over DE cells) (FIG.12C-12D) Sequential up and downregulation of AFE-specific proteinsduring differentiation of iPSCs to AETII quantified by qRT-PCR in (FIG.12C) C1 clone and (FIG. 12D) C2 clone. Data expressed as quantificationof mRNA normalized to GAPDH and average fold change in gene expressionover AFE cells; bar indicate ±SEM and n=3 independent experiments.

FIG. 13, comprising FIG. 13A through FIG. 13D, is a series of imagesdepicting the kinetics of NKX2.1 and SPC expression duringdifferentiation of iPSC cells (C1 clone) to lung alveolar epithelium.(FIG. 13A-13B) Kinetics of NKX2.1 and SPC mRNA expression at differentdays, quantified by real time qRT-PCR. (FIG. 13A) SPC expression duringdifferentiation of iPS cells to AETII. Ct values of SPC is normalized toGAPDH and expressed to levels seen in ATII cells isolated from humanlung (hATII) (FIG. 13B) NKX2.1 during differentiation of iPS cells toAETII. Data expressed as quantification of mRNA normalized to GAPDH andexpressed to the level of seen in isolated human primary type II. (FIG.13C-13D) Flow cytometry analysis for the percentage of positive cellsfor (FIG. 13C) NKX2.1 from day 13 to day 22 and (FIG. 13D) SPC from day10 to day 22. Bars indicate ±SEM and n=3 independent experiments for PCRand flow cytometry.

FIG. 14, comprising FIG. 14A through FIG. 14D, is a series of imagesdepicting the kinetics of NKX2.1 and SPC expression duringdifferentiation of iPSC cells (C2 clone) to lung alveolar epithelium.(FIG. 14A-14B) NKX2.1 and SPC mRNA expression at different days,quantified by real time RT-PCR in iPSC-derived AETII (C2 clone). (FIG.14A) SPC expression during differentiation from day 0 to day 32. Ctvalue for SPC is normalized to GAPDH and expressed to levels seen inAETII cells isolated from human lung (hATII) (FIG. 14B) NKX2.1expression during differentiation. Data expressed as quantification ofmRNA normalized to GAPDH and expressed to the levels seen in isolatedhuman type II (FIG. 14C-14D) Flow cytometry analysis for the percentageof positive cells for (FIG. 14C) NKX2.1 from day 13 to day 22 and SPC(FIG. 14D) from day 10 to day 22. Bars indicate ±SEM and n=3 independentexperiments for PCR and flow cytometry.

FIG. 15, comprising FIG. 15A through FIG. 15D, is a series of imagesdepicting the kinetics of α6β4 and CD166 expression duringdifferentiation of iPSC cells to lung alveolar epithelium. (FIG. 15 andFIG. 15C) Flow cytometry analysis for the percentage of positive cellsfor CD166 from day 10 to day 28 in AETII derived from (FIG. 15A) C1clone and (FIG. 15C) C2 clone. (FIG. 15B and FIG. 15D) Flow cytometryanalysis for the percentage of positive cells for α6β4 from day 8 to day22 in AETII derived from (FIG. 15B) C1 clone and (FIG. 15D) C2 clone.

FIG. 16, comprising FIG. 16A through FIG. 16E, is a series of imagesdepicting the pluripotency marker analysis in C2 clone in day 0 andduring differentiation to AETII. (FIG. 16A). Immunofluorescent stainingof iPSC markers; OCT4, Nanog, SSEA4, Tra1-81 in both iPSC clone C1 andC2 (Scale bar, 100 μm). Both clones were positive for pluripotency genesat day 0. (FIG. 16B-C) Downregulation of iPSC-specific genes OCT4, SOX2,and Nanog during differentiation to AETI. The expression of OCT4, SOX2,and Nanog were downregulated over the time and by day 32 these markerswere undetectable in iPSC-derived AETII derived from both (FIG. 16B)clone C1 and (FIG. 16C) clone C2 (Data expressed as quantification ofmRNA normalized to GAPDH and average fold change in gene expression overiPS cells at day 0, bar indicates SEM and n=3 independent experiments),(FIG. 16D-16E) Expression of OCT4 and SPC in differentiated AETII cellson day 0 compare to day 22 analyzed by flow cytometry for (FIG. 16D) C1clone and (FIG. 16E) C2 clone. At day 22 of differentiation, SPCpositive iPSC-derived AETII cells were negative for OCT4.

FIG. 17, comprising FIG. 17A through FIG. 17M, is a series of imagesdemonstrating that AETII derived from iPSC C2 clone respond torecellularize 3D lung tissue scaffolds in bioreactor (FIG. 17A-17C) H&Estaining of seeded rat lung scaffold with iPSC-derived AETII cells at(FIG. 17A) day 3 and (FIG. 17B-17C) at day 7 in bioreactor. Scale bar,200 μm. (FIG. 17D-17F) Immunostaining for SPC on seeded rat lungscaffold with iPSC-derived AETII cells cultured in bioreactor at day 7(FIG. 17D) Nuclei were stained with DAPI; (FIG. 17E) shows Pro-SPCstaining (FIG. 17F) Merge, Scale bar, 50 μm (FIG. 17G-17I)Immunostaining for NKX2.1 on seeded rat lung scaffold with iPSC-derivedAETII cells cultured in bioreactor at day 7 (FIG. 17G) Nuclei werestained with DAPI; (FIG. 17H) shows NKX2.1 staining (FIG. 17I) Merge,Scale bar, 50 μm (FIG. 17J-17M) Immunostaining for PCNA and caspase ofbioreactor cultured iPSC-derived APTII cells at day 7. Scale bar, 49 μm

DETAILED DESCRIPTION

The invention provides a method of differentiating a stem cell into alung cell, preferably an alveolar epithelial type II cell. In oneembodiment, the stem cell is a human induced pluripotent stem (iPS)cell.

In one embodiment, the method of generating a lung cell comprisesculturing a stem cell without serum in the presence of Activin A inorder to generate a definitive endoderm (DE) population. The DEpopulation can then be differentiated into anterior foregut endoderm(AFE) by culturing the DE cells in the presence of the combination of anextracellular matrix (ECM) protein and a culture medium supplementedwith an inhibitor of BMP and an inhibitor of TGF-β signaling.Preferably, the inhibitor of BMP is NOGGIN and the inhibitor of TGF-βsignaling is SB-431542.

The AFE cells can be cultured in the presence of a differentiationmedium to induce differentiation into a desired cell type. For example,induction to differentiate into an alveolar epithelial type II cellcomprises culturing the AFE cells in the presence of a differentiationmedium comprising FGF-10, EGF, Wnt3a, and KGF. Preferably, the alveolarepithelial type II cell differentiation medium does not include BMP4.

The compositions and methods of the invention are useful for among otherthings, drug discovery, toxicity testing, disease pathology,investigating lung developmental biology, and the like.

The invention relates to the discovery that alveolar epithelial type IIcells can be generated in vitro. Accordingly, the invention providesmethods and compositions for the generation of alveolar epithelial typeII cells as a form of regenerative medicine. In one embodiment, themethod allows for the generation of a pure population of alveolar typeII progenitor cells whereby the cells express a high percentage of lungalveolar type II markers including but is not limited to SPC, SPB,Mucin-1, and CD54.

The invention also provides a method of alleviating or treating a lungdefect in a mammal, preferably a human. The method comprisesadministering to the mammal in need thereof a therapeutically effectiveamount of a composition comprising an alveolar epithelial type II cellor otherwise cells that exhibit at least one characteristic of analveolar epithelial type II cell, thereby alleviating or treating thelung defect in the mammal.

In one embodiment, the invention provides a method of repairing injuredor diseased alveolar epithelial tissue in the lung of a mammalcomprising transplanting into the lung, at a site comprising injured ordiseased alveolar epithelial tissue, a population of differentiated stemcells, or progeny thereof, at least 95%, preferably at least 96%, morepreferably at least 97%, more preferably 98%, yet more preferably atleast 99% of which exhibit an alveolar type II phenotype. The populationof cells with alveolar type II phenotype is prepared using the methodsof the invention, and, after transplantation, is effective to repair atleast a portion of the injured or diseased alveolar epithelial tissue atthe site.

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry, and nucleic acidchemistry and hybridization are those well-known and commonly employedin the art.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” will be understood by persons of ordinary skill in theart and will vary to some extent based on the context in which it isused.

The term “adult stem cell” or “ASC” is used to refer to any multipotentstem cell derived from non-embryonic tissue, including fetal, juvenile,and adult tissue. Stem cells have been isolated from a wide variety ofadult tissues including blood, bone marrow, brain, olfactory epithelium,skin, pancreas, skeletal muscle, and cardiac muscle. Each of these stemcells can be characterized based on gene expression, factorresponsiveness, and morphology in culture. Exemplary adult stem cellsinclude neural stem cells, neural crest stem cells, mesenchymal stemcells, hematopoietic stem cells, and pancreatic stem cells. As indicatedabove, stem cells have been found resident in virtually every tissue.Accordingly, the present invention appreciates that stem cellpopulations can be isolated from virtually any animal tissue.

As used herein, “anterior foregut endoderm” refers to endoderm that isanterior to the endoderm that gives rise to the liver. One of ordinaryskill in the art will readily appreciate that “anterior foregutendoderm” thus includes, for example, pharyngeal endoderm and other,more highly differentiated populations of endodermal cells and that thevarious cell types encompassed by the term “anterior foregut endoderm”may exhibit different expression patterns of molecular markers. One ofordinary skill in the art will appreciate that “anterior foregutendoderm” gives rise to various tissues, e.g., tonsils, tympanicmembrane, thyroid, parathyroid glands, thymus, trachea, esophagus,stomach, lung and larynx/pharynx.

As used herein, “autologous” refers to a biological material derivedfrom the same individual into whom the material will later bere-introduced.

As used herein, “allogeneic” refers to a biological material derivedfrom a genetically different individual of the same species as theindividual into whom the material will be introduced.

As used here, “biocompatible” refers to any material, which, whenimplanted in a mammal, does not provoke an adverse response in themammal. A biocompatible material, when introduced into an individual, isnot toxic or injurious to that individual, nor does it induceimmunological rejection of the material in the mammal.

As used herein, “anterior foregut endoderm” refers to endoderm that isanterior to the endoderm. One of ordinary skill in the art will readilyappreciate that “anterior foregut endoderm” thus includes, for example,pharyngeal endoderm and other, more highly differentiated populations ofendodermal cells and that the various cell types encompassed by the term“anterior foregut endoderm” may exhibit different expression patterns ofmolecular markers. One of ordinary skill in the art will appreciate that“anterior foregut endoderm” gives rise to various tissues, e.g.,tonsils, tympanic membrane, thyroid, parathyroid glands, thymus,trachea, esophagus, stomach, lung and larynx/pharynx.

As used herein, to “alleviate” a disease, defect, disorder or conditionmeans reducing the severity of one or more symptoms of the disease,defect, disorder or condition.

As used herein, the term “basal medium” refers to a solution of aminoacids, vitamins, salts, and nutrients that is effective to support thegrowth of cells in culture, although normally these compounds will notsupport cell growth unless supplemented with additional compounds. Thenutrients include a carbon source (e.g., a sugar such as glucose) thatcan be metabolized by the cells, as well as other compounds necessaryfor the cells' survival. These are compounds that the cells themselvescannot synthesize, due to the absence of one or more of the gene(s) thatencode the protein(s) necessary to synthesize the compound (e.g.,essential amino acids) or, with respect to compounds which the cells cansynthesize, because of their particular developmental state the gene(s)encoding the necessary biosynthetic proteins are not being expressed assufficient levels. A number of base media are known in the art ofmammalian cell culture, such as Dulbecco's Modified Eagle Media (DMEM),Knockout-DMEM (KO-DMEM), and DMEM/F12, although any base medium thatsupports the growth of primate embryonic stem cells in a substantiallyundifferentiated state can be employed. As used here, “biocompatible”refers to any material, which, when implanted in a mammal, does notprovoke an adverse response in the mammal. A biocompatible material,when introduced into an individual, is not toxic or injurious to thatindividual, nor does it induce immunological rejection of the materialin the mammal.

As used herein, the term “biocompatible lattice,” is meant to refer to asubstrate that can facilitate formation into three-dimensionalstructures conducive for tissue development. Thus, for example, cellscan be cultured or seeded onto such a biocompatible lattice, such as onethat includes extracellular matrix material, synthetic polymers,cytokines, growth factors, etc. The lattice can be molded into desiredshapes for facilitating the development of tissue types. Also, at leastat an early stage during culturing of the cells, the medium and/orsubstrate is supplemented with factors (e.g., growth factors, cytokines,extracellular matrix material, etc.) that facilitate the development ofappropriate tissue types and structures.

“Bioactive agents,” as used herein, can include one or more of thefollowing: chemotactic agents; therapeutic agents (e.g., antibiotics,steroidal and non-steroidal analgesics and anti-inflammatories(including certain amino acids such as glycine), anti-rejection agentssuch as immunosuppressants and anti-cancer drugs); various proteins(e.g., short term peptides, bone morphogenic proteins, collagen,hyaluronic acid, glycoproteins, and lipoprotein); cell attachmentmediators; biologically active ligands; integrin binding sequence;ligands; various growth and/or differentiation agents and fragmentsthereof (e.g., epidermal growth factor (EGF), hepatocyte growth factor(HGF), vascular endothelial growth factors (VEGF), fibroblast growthfactors (e.g., bFGF), platelet derived growth factors (PDGF), insulinderived growth factor (e.g., IGF-1, IGF-II) and transforming growthfactors (e.g., TGFβ I-III), parathyroid hormone, parathyroid hormonerelated peptide, bone morphogenic proteins (e.g., BMP-2, BMP-4; BMP-6;BMP-7; BMP-12; BMP-13; BMP-14), sonic hedgehog, growth differentiationfactors (e.g., GDFS, GDF6, GDF8), recombinant human growth factors(e.g., MP52, and MP-52 variant rhGDF-5), cartilage-derived morphogenicproteins (CDMP-1; CDMP-2, CDMP-3)); small molecules that affect theupregulation of specific growth factors; tenascin-C; hyaluronic acid;chondroitin sulfate; fibronectin; decorin; thromboelastin;thrombin-derived peptides; heparin-binding domains; heparin; heparansulfate. Suitable effectors likewise include the agonists andantagonists of the agents described above. The growth factor can alsoinclude combinations of the growth factors described above. In addition,the growth factor can be autologous growth factor that is supplied byplatelets in the blood. In this case, the growth factor from plateletswill be an undefined cocktail of various growth factors. If other suchsubstances have therapeutic value in the orthopedic field, it isanticipated that at least some of these substances will have use in thepresent invention, and such substances should be included in the meaningof “bioactive agent” and “bioactive agents” unless expressly limitedotherwise. Preferred examples of bioactive agents include culture media,bone morphogenic proteins, growth factors, growth differentiationfactors, recombinant human growth factors, cartilage-derived morphogenicproteins, hydrogels, polymers, antibiotics, anti-inflammatorymedications, immunosuppressive mediations, autologous, allogenic orxenologous cells such as stem cells, chondrocytes, fibroblast andproteins such as collagen and hyaluronic acid. Bioactive agents can beautologous, allogenic, xenogenic or recombinant.

The term “biologically compatible carrier” or “biologically compatiblemedium” refers to reagents, cells, compounds, materials, compositions,and/or dosage formulations which are suitable for use in contact withthe tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other complication commensurate with areasonable benefit/risk ratio.

The terms “cells” and “population of cells” are used interchangeably andrefer to a plurality of cells, i.e., more than one cell. The populationmay be a pure population comprising one cell type. Alternatively, thepopulation may comprise more than one cell type. In the presentinvention, there is no limit on the number of cell types that a cellpopulation may comprise.

The term “cell medium” as used herein, refers to a medium useful forculturing cells. An example of a cell medium is a medium comprisingDMEM/F 12 Ham's, 10% fetal bovine serum, 100 U penicillin/100 μgstreptomycin/0.25 μg Fungizone. Typically, the cell medium comprises abase medium, serum and an antibiotic/antimycotic. However, cells can becultured with stromal cell medium without an antibiotic/antimycotic andsupplemented with at least one growth factor. Preferably the growthfactor is human epidermal growth factor (hEGF). The preferredconcentration of hEGF is about 1-50 ng/ml, more preferably theconcentration is about 5 ng/ml. The preferred base medium is DMEM/F12(1:1). The preferred serum is fetal bovine serum (FBS) but other seramay be used including horse serum or human serum. Preferably up to 20%FBS will be added to the above media in order to support the growth ofstromal cells. However, a defined medium could be used if the necessarygrowth factors, cytokines, and hormones in FBS for cell growth areidentified and provided at appropriate concentrations in the growthmedium. It is further recognized that additional components may be addedto the culture medium. Such components include but are not limited toantibiotics, antimycotics, albumin, growth factors, amino acids, andother components known to the art for the culture of cells. Antibioticswhich can be added into the medium include, but are not limited to,penicillin and streptomycin. The concentration of penicillin in theculture medium is about 10 to about 200 units per ml. The concentrationof streptomycin in the culture medium is about 10 to about 200 μg/ml.However, the invention should in no way be construed to be limited toany one medium for culturing cells. Rather, any media capable ofsupporting cells in tissue culture may be used.

The term “decellularized” or “decellularization” as used herein refersto a biostructure (e.g., an organ, or part of an organ), from which thecellular and tissue content has been removed leaving behind an intactacellular infra-structure. Organs such as the kidney are composed ofvarious specialized tissues. The specialized tissue structures of anorgan, or parenchyma, provide the specific function associated with theorgan. The supporting fibrous network of the organ is the stroma. Mostorgans have a stromal framework composed of unspecialized connectingtissue which supports the specialized tissue. The process ofdecellularization removes the specialized tissue, leaving behind thecomplex three-dimensional network of connective tissue. The connectivetissue infra-structure is primarily composed of collagen. Thedecellularized structure provides a biocompatible substrate onto whichdifferent cell populations can be infused. Decellularized biostructurescan be rigid, or semi-rigid, having an ability to alter their shapes.Examples of decellularized organs useful in the present inventioninclude, but are not limited to, the heart, lung, kidney, liver,pancreas, spleen, bladder, ureter and urethra, cartilage, bone, brain,spine cord, peripheral nerve.

As used herein “definitive endoderm (DE)” and definitive endoderm cells(DE-cells) refers to cells exhibiting such as but not limited to proteinor gene expression and or/or morphology typical to cells of thedefinitive endoderm or a composition comprising a significant number ofcells resembling the cells of the definitive endoderm. A definitiveendoderm cell can expresses the marker Sox17. Other markers ofdefinitive endoderm cells include, but are not limited to MIXL2, GATA4,HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1. Definitiveendoderm cells have the capacity to differentiate into cells includingthose of the liver, lung, pancreas, thymus, intestine, stomach andthyroid.

The term “dedifferentiation”, as used herein, refers to the return of acell to a less specialized state. After dedifferentiation, such a cellwill have the capacity to differentiate into more or different celltypes than was possible prior to re-programming. The process of reversedifferentiation (i.e., de-differentiation) is likely more complicatedthan differentiation and requires “re-programming” the cell to becomemore primitive.

The term “differentiated cell” is meant any primary cell that is not, inits native form, pluripotent as that term is defined herein. Statedanother way, the term “differentiated cell” refers to a cell of a morespecialized cell type derived from a cell of a less specialized celltype (e.g., a stem cell such as an induced pluripotent stem cell) in acellular differentiation process. Without wishing to be limited totheory, a pluripotent stem cell in the course of normal ontogeny candifferentiate first to an endoderm cell that is capable of forming lungcells and other endoderm cell types. Endoderm cells can also bedifferentiate into other cells of endodermal origin, e.g. lung, liver,intestine, thymus etc.

“Differentiation medium” is used herein to refer to a cell growth mediumcomprising an additive or a lack of an additive such that a stem cell,fetal pulmonary cell or other such progenitor cell, that is not fullydifferentiated, develops into a cell with some or all of thecharacteristics of a differentiated cell when incubated in the medium.

The term “embryonic stem cell” is used to refer to the pluripotent stemcells of the inner cell mass of the embryonic blastocyst (see U.S. Pat.Nos. 5,843,780, 6,200,806). Such cells can similarly be obtained fromthe inner cell mass of blastocysts derived from somatic cell nucleartransfer (see, for example, U.S. Pat. Nos. 5,945,577, 5,994,619,6,235,970). The distinguishing characteristics of an embryonic stem celldefine an embryonic stem cell phenotype. Accordingly, a cell has thephenotype of an embryonic stem cell if it possesses one or more of theunique characteristics of an embryonic stem cell such that that cell canbe distinguished from other cells. Exemplary distinguishing embryonicstem cell characteristics include, without limitation, gene expressionprofile, proliferative capacity, differentiation capacity, karyotype,responsiveness to particular culture conditions, and the like.

As used herein, “epithelial cell” means a cell which forms the outersurface of the body and lines organs, cavities and mucosal surfaces.

As used herein, “endothelial cell” means a cell which lines the bloodand lymphatic vessels and various other body cavities.

As used herein “endogenous” refers to any material from or producedinside an organism, cell or system.

“Exogenous” refers to any material introduced into or produced outsidean organism, cell, or system.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

The term “endoderm cell” as used herein refers to a cell which is fromone of the three primary germ cell layers in the very early embryo (theother two germ cell layers are the mesoderm and ectoderm). The endodermis the innermost of the three layers. An endoderm cell differentiates togive rise first to the embryonic gut and then to the linings ofrespiratory and digestive tracts (e.g. the intestine), the liver and thepancreas.

“Expandability” is used herein to refer to the capacity of a cell toproliferate, for example, to expand in number or, in the case of apopulation of cells, to undergo population doublings.

As used herein, “extracellular matrix composition” includes both solubleand non-soluble fractions or any portion thereof. The non-solublefraction includes those secreted ECM proteins and biological componentsthat are deposited on the support or scaffold. The soluble fractionincludes refers to culture media in which cells have been cultured andinto which the cells have secreted active agent(s) and includes thoseproteins and biological components not deposited on the scaffold. Bothfractions may be collected, and optionally further processed, and usedindividually or in combination in a variety of applications as describedherein.

As used herein, a “fetal pulmonary cells” (FPCs) refer to cells isolatedfrom the lung tissue of an embryo. A mixed population of FPCs caninclude, but is not limited to epithelial, mesenchymal, and endothelialcells.

As used herein, a “graft” refers to a cell, tissue or organ that isimplanted into an individual, typically to replace, correct or otherwiseovercome a defect. A graft may further comprise a scaffold. The tissueor organ may consist of cells that originate from the same individual;this graft is referred to herein by the following interchangeable terms:“autograft,” “autologous transplant,” “autologous implant” and“autologous graft.” A graft comprising cells from a geneticallydifferent individual of the same species is referred to herein by thefollowing interchangeable terms: “allograft”, “allogeneic transplant,”“allogeneic implant” and “allogeneic graft.” A graft from an individualto his identical twin is referred to herein as an “isograft”, a“syngeneic transplant”, a “syngeneic implant” or a “syngeneic graft.” A“xenograft,” “xenogeneic transplant” or “xenogeneic implant” refers to agraft from one individual to another of a different species.

As used herein, the term “growth factor product” refers to a protein,peptide, mitogen, or other molecule having a growth, proliferative,differentiative, or trophic effect on a cell. Growth factors include,but are not limited to, fibroblast growth factor (FGF), basic fibroblastgrowth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermalgrowth factor (EGF), insulin-like growth factor-I (IGF-T), insulin-likegrowth factor-II (IGF-II), platelet-derived growth factor (PDGF),vascular endothelial cell growth factor (VEGF), activin-A, bonemorphogenic proteins (BMPs), insulin, growth hormone, erythropoietin,thrombopoietin, interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin7 (IL-7), macrophage colony stimulating factor, c-kit ligand/stem cellfactor, osteoprotegerin ligand, insulin, nerve growth factor, ciliaryneurotrophic factor, cytokines, chemokines, morphogens, neutralizingantibodies, other proteins, and small molecules. Preferably, the FGF isselected from the group selected from FGF2, FGF7, FGF10, and anycombination thereof.

As used herein, the term “growth medium” is meant to refer to a culturemedium that promotes growth of cells. A growth medium will generallycontain animal serum. In some instances, the growth medium may notcontain animal serum.

As used herein, “human pluripotent stem cells” (hPS) refers to cellsthat may be derived from any source and that are capable, underappropriate conditions, of producing human progeny of different celltypes that are derivatives of all of the 3 germinal layers (endoderm,mesoderm, and ectoderm). hPS cells may have the ability to form ateratoma in 8-12 week old SCID mice and/or the ability to formidentifiable cells of all three germ layers in tissue culture. Includedin the definition of human pluripotent stem cells are embryonic cells ofvarious types including human blastocyst derived stem (hBS) cells inliterature often denoted as human embryonic stem (hES) cells, (see,e.g., Thomson et al. (1998), Heins et. al. (2004), as well as inducedpluripotent stem cells (see, e.g. Yu et al., (2007) Science 318:5858);Takahashi et al., (2007) Cell 131(5):861). The various methods and otherembodiments described herein may require or utilize hPS cells from avariety of sources. For example, hPS cells suitable for use may beobtained from developing embryos. Additionally or alternatively,suitable hPS cells may be obtained from established cell lines and/orhuman induced pluripotent stem (hiPS) cells.

As used herein “hiPS cells” refers to human induced pluripotent stemcells.

An “isolated cell” refers to a cell which has been separated from othercomponents and/or cells which naturally accompany the isolated cell in atissue or mammal.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in anaturally-occurring state, i.e., a DNA fragment which has been removedfrom the sequences which are normally adjacent to the fragment, i.e.,the sequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, i.e., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (i.e.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

The term “lung specific” refers to a nucleic acid molecule orpolypeptide that is expressed predominantly in the lung as compared toother tissues in the body. In a preferred embodiment, a “lung specific”nucleic acid molecule or polypeptide is expressed at a level that is5-fold higher than any other tissue in the body. In a more preferredembodiment, the “lung specific” nucleic acid molecule or polypeptide isexpressed at a level that is 10-fold higher than any other tissue in thebody, more preferably at least 15-fold, 20-fold, 25-fold, 50-fold or100-fold higher than any other tissue in the body. Nucleic acid moleculelevels may be measured by nucleic acid hybridization, such as Northernblot hybridization, or quantitative PCR. Polypeptide levels may bemeasured by any method known to accurately measure protein levels, suchas Western blot analysis.

“Lung tissue” can include, but is not limited to, all lung tissuestructures and associated tissues, including, but not limited to, veins,arteries, vessels, capillaries, and cells of the type that are part of,or associated with, such structures; lung and pleural tissue; andvascular smooth muscle, pericyte, and vascular endothelial lineagesand/or phenotypes.

The terms “precursor cell,” “progenitor cell,” and “stem cell” are usedinterchangeably in the art and as used herein refer either to apluripotent or lineage-uncommitted progenitor cell, which is potentiallycapable of an unlimited number of mitotic divisions to either renewitself or to produce progeny cells which will differentiate into thedesired cell type. In contrast to pluripotent stem cells,lineage-committed progenitor cells are generally considered to beincapable of giving rise to numerous cell types that phenotypicallydiffer from each other. Instead, progenitor cells give rise to one orpossibly two lineage-committed cell types.

“Proliferation” is used herein to refer to the reproduction ormultiplication of similar forms, especially of cells. That is,proliferation encompasses production of a greater number of cells, andcan be measured by, among other things, simply counting the numbers ofcells, measuring incorporation of ³H-thymidine into the cell, and thelike.

“Progression of or through the cell cycle” is used herein to refer tothe process by which a cell prepares for and/or enters mitosis and/ormeiosis. Progression through the cell cycle includes progression throughthe G1 phase, the S phase, the G2 phase, and the M-phase.

As used herein, “scaffold” refers to a structure, comprising abiocompatible material, that provides a surface suitable for adherenceand proliferation of cells. A scaffold may further provide mechanicalstability and support. A scaffold may be in a particular shape or formso as to influence or delimit a three-dimensional shape or form assumedby a population of proliferating cells. Such shapes or forms include,but are not limited to, films (e.g. a form with two-dimensionssubstantially greater than the third dimension), ribbons, cords, sheets,flat discs, cylinders, spheres, 3-dimensional amorphous shapes, etc.

As used herein, the phrase “stem cells” refers both to the earliestrenewable cell population responsible for generating cell mass in atissue or body and the very early progenitor cells, which are somewhatmore differentiated, yet are not committed and can readily revert tobecome a part of the earliest renewable cell population.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. Thus, a substantially purifiedcell refers to a cell which has been purified from other cell types withwhich it is normally associated in its naturally-occurring state.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, a subject is preferably a mammal suchas a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) anda primate (e.g., monkey and human), most preferably a human.

As used herein, to “treat” means reducing the frequency with whichsymptoms of a disease, defect, disorder, or adverse condition, and thelike, are experienced by a patient.

As used herein, a “therapeutically effective amount” is the amount of acomposition of the invention sufficient to provide a beneficial effectto the individual to whom the composition is administered.

As used herein, “tissue engineering” refers to the process of generatingtissues ex vivo for use in tissue replacement or reconstruction. Tissueengineering is an example of “regenerative medicine,” which encompassesapproaches to the repair or replacement of tissues and organs byincorporation of cells, gene or other biological building blocks, alongwith bioengineered materials and technologies.

As used herein, the terms “tissue grafting” and “tissue reconstructing”both refer to implanting a graft into an individual to treat oralleviate a tissue defect, such as a lung defect or a soft tissuedefect.

“Transplant” refers to a biocompatible lattice or a donor tissue, organor cell, to be transplanted. An example of a transplant may include butis not limited to skin cells or tissue, bone marrow, and solid organssuch as heart, pancreas, kidney, lung and liver.

Generally, a “trophic factor” is defined as a substance that promotessurvival, growth, proliferation and/or maturation of a cell, orstimulates increased activity of a cell.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

The present invention relates to the successful generation of a purepopulation of alveolar epithelial type II cells from stem cells. Thealveolar epithelial type II cells differentiated from stem cells serveas a promising source of cells for use therapeutically to treat distallung diseases, lung injuries, and genetic diseases that affect the lung.

The present invention provides a method of differentiating stem cellsinto cells that exhibit at least one characteristic of an alveolarepithelial type II cell. In one embodiment, the method comprises twophases wherein the first phase comprises generating a definitiveendoderm (DE) population from a stem cell population and the secondphase comprising differentiating the DE population into cells of theanterior foregut endoderm (AFE). With respect to the first phase, in oneembodiment, stem cells are initially cultured without serum in thepresence of Activin A in order to generate a DE population. With respectto the second phase, in one embodiment, the DE population is cultured inthe presence of a human extracellular matrix (ECM) protein incombination with a culture medium supplemented with an inhibitor of BMPand an inhibitor of TGF-β signaling and in the absence of Activin A inorder to induce differentiation into AFE. Preferably, the inhibitor ofBMP is NOGGIN and the inhibitor of TGF-β signaling is SB-431542.

After AFE cells are generated, they can be cultured in the presence of adifferentiation medium to induce differentiation into a desired celltype. For example, the AFE cells can be induced to differentiation intoa cell that exhibits at least one characteristic of an alveolarepithelial type II cell wherein the differentiation medium comprisesFGF-10, EGF, Wnt3a, and KGF. Preferably, the alveolar epithelial type IIcell differentiation medium does not include BMP4.

Accordingly, the invention provides a method for directeddifferentiation of stem cells into cell types and tissues derived fromthe AFE including but is not limited to alveolar type II cells.

The invention is partly based upon the discovery that the uniqueprotocol disclosed elsewhere herein allows for the highly efficientconversion of AFE cells into alveolar type II progenitor cells. Forexample, the use of ECM protein-coated surfaces in combination withspecific growth factors generated a cell population that expressed ahigh percentage of lung alveolar type II cell markers. In one example,the methods of the invention allows for the generation of a purepopulation of alveolar type II cells derived from human stem cells,preferably human induced pluripotent stem (iPS) cells.

In one embodiment, the method of differentiating stem cells towards analveolar type II cell phenotype is distinct from prior art in that thestep of differentiating AFE to an alveolar type II cell phenotyperequires the use of the combination of growth factors and ECM proteins.In one embodiment, the combination of growth factors includes FGF-10,EGF, Wnt3a, and KGF. In another embodiment, the combination of growthfactors excludes BMP4. With respect to ECM proteins, the cells in oneembodiment, can be cultured in the presence of ECM proteins that mimicECM proteins that exist during embryogenesis for lung development (e.g.,collagens, laminin, fibronectin, tenascin, elastin, and a number ofproteoglycans and glycosaminoglycans).

Definitive Endoderm

During embryonic development, the tissues of the body are formed fromthree major cell populations: ectoderm, mesoderm and definitiveendoderm. These cell populations, also known as primary germ celllayers, are formed through a process known as gastrulation. Followinggastrulation, each primary germ cell layer generates a specific set ofcell populations and tissues. Mesoderm gives rise to, for example, bloodcells, endothelial cells, cardiac and skeletal muscle, and adipocytes.Definitive endoderm generates, for example, liver, pancreas and lung.Ectoderm gives rise to, for example, the nervous system, skin andadrenal tissues.

Described herein is the demonstration that definitive endoderm can beproduced, purified and expanded from stem cells. Thus purified orsub-cultured stem cell-derived definitive endoderm can be used as aplatform for differentiation toward lung progenitors (e.g., alveolartype II progenitor cells). In one embodiment, the invention provides amethod of generating cell populations comprising definitive endodermcells from a cell population substantially initially consistingessentially of stem cells, preferably human induced pluripotent stem(iPS) cells.

In one embodiment, the invention provides a method of producing endodermcells, such as definitive endoderm cells by exposing stem cells such asembryonic stem cells (ES) or iPS cells to an effective amount of atleast one compound described herein to differentiate the stem cells intothe endoderm cells such as definitive endoderm cells. Differentiatedendoderm cells produced by the methods disclosed herein can bedifferentiated into endoderm derivatives such as pancreas, thymus,liver, stomach, intestine and lung. Another aspect of the presentinvention relates to a method of producing alveolar type II progenitorcells by exposing endoderm cells, such as definitive endoderm cells toan effective amount of at least one compound described herein todifferentiate the definitive endoderm cells into alveolar type IIprogenitor cells.

Methods of the invention can be used for stimulating differentiation ofstem cells into DE cells in medium which is free of serum and free ofserum extract. Preferably, such methods are also carried out in theabsence of feeder cells and/or feeder cell extracts.

For example, differentiation of stem cells into DE cells can be carriedout comprising the steps of: 1) maintaining stem cells in a firstculture medium for a period of time, optionally on feeders, in thepresence of serum or an extract of serum or in a serum free/serumextract free medium; 2) replacing the first medium with a second serumfree medium comprising activin or removing the serum or the serumextract from the first medium and withdrawing the feeders (if present)and adding activin, so that the first medium is free of feeders, serumand serum extract; and 3) subsequently propagating the stem cells in themedium comprising activin in order to obtain cells comprising DE cells.

In one embodiment, directed differentiation of pluripotent cells, e.g.,stem cells, into definitive endoderm can be obtained by application ofhigh concentrations of Activin A. Without wishing to be bound by anyparticular theory, it is believed that the scientific basis for thisstrategy is that signaling by the morphogen nodal is required forendoderm formation. Activin A activates the same receptor as nodal, butis available as a soluble cytokine.

In one embodiment, generation of definitive endoderm stem cells may beaccomplished by adapting a protocol used to develop definitive endodermfrom mouse ES cells (Kubo et al, 2004 Development 131: 1651-1662).Preferably, the stem cells are initially cultured without serum in thepresence of a high concentration of Activin A (about 50-500 ng/ml, about75-150 ng/ml, about 100 ng/ml).

One can use any means common to one of ordinary skill in the art toconfirm the presence of an endoderm cell, e.g. a definitive endodermcell produced the methods of the invention. In some embodiments, thepresence of endoderm cells can be detected using suitable markers suchas those listed in U.S. Pat. No. 7,326,572, which is incorporated hereinby reference.

In some embodiments, the presence of definitive endoderm markers, e.g.chemically induced definitive endoderm cells, can be evaluated bydetecting the presence or absence of one or more markers indicative of adefinitive endoderm cell. In some embodiments, the method can includedetecting the positive expression (e.g., the presence) of a marker fordefinitive endoderm cells. In some embodiments, the marker can bedetected using a reagent, e.g., a reagent for the detection of one ormore of SOX17, HNF3β (Fox2A), MIXL2, GATA4, GSC, FGF17, VWF, CALCR,FOXQ1, CMKOR1, CRIP1, and the like. In particular, definitive endodermcells express Sox17 and/or HNF3B, and do not express significant levelsof extra-embryonic endoderm markers such as GATA4, SPARC, APF, and DAB.Other positive markers for definitive endoderm cells also include butare not limited to Nodal, Tmprss2, Tmem30b, St14, Spink3, Sh3g12, Ripk4,Rab15, Npnt, Clic6, Cldn8, Cacna1b, Bnip1, Anxa4, Emb, FoxA1, andRbm35a.

Negative markers (e.g., the absence of significant levels of expression)for definitive endoderm cells include extra-embryonic (EE) endodermmarkers such as Gata4, SPARC, APF and DAB, as well as negative markersZic, Pax6, Flk1 or CD31. Negative markers of definitive endoderm cellsare useful for the purposes of negative selection of non-definitiveendoderm cells (e.g., selection and discarding cells which expressGata4, SPARC, APF, DAB, Zic, Pax6, Flk1 or CD31) or for identificationof cells which do not express these negative markers (e.g. definitiveendoderm cells).

A reagent for a marker can be, for example, an antibody against themarker or primers for a RT-PCR or PCR reaction, e.g., asemi-quantitative or quantitative RT-PCR or PCR reaction. Such markerscan be used to evaluate whether a definitive endoderm cell has beenproduced. The antibody or other detection reagent can be linked to alabel, e.g., a radiological, fluorescent (e.g., GFP) or colorimetriclabel for use in detection. If the detection reagent is a primer, it canbe supplied in dry preparation, e.g., lyophilized, or in a solution.

The progression of a pluripotent stem cell to a definitive endoderm canbe monitored by determining the expression of markers characteristic ofdefinitive endoderm cells. In some processes, the expression of certainmarkers is determined by detecting the presence or absence of themarker. Alternatively, the expression of certain markers can bedetermined by measuring the level at which the marker is present in thecells of the cell culture or cell population. In certain processes, theexpression of markers characteristic of definitive endoderm cells aswell as the lack of significant expression of markers characteristic ofthe pluripotent stem cell from which it was derived is determined.

Anterior Foregut Endoderm (AFE)

Lung, esophagus and trachea are derived from anterior foregut endodermdistal to the pouches. The ability to generate populations of anteriorforegut/pharyngeal endoderm cells from pluripotent cells would be usefulin cell replacement therapy for these tissues, in assays for agents thataffect cell growth and differentiation, and in studies on tissuedevelopment and differentiation.

The definitive endoderm cells of the present invention may be allowed todifferentiate into cells of the anterior foregut endoderm. It has beendiscovered that although Activin A induces endoderm, Activin Aposteriorizes this tissue. Preparation of anterior foregut endoderm thusrequires reducing or removing Activin A following formation ofdefinitive endoderm. In certain aspects, the present invention thusprovides a method for the formation of a population of cells enrichedfor anterior foregut endoderm, and the depletion of mid- and posteriorendoderm signals.

In one embodiment, the definitive endoderm cell population can then bedifferentiated into anterior foregut endoderm by culturing thedefinitive endoderm cells in the presence of the combination of anextracellular matrix (ECM) protein and a culture medium supplementedwith an inhibitor of BMP and an inhibitor of TGF-β signaling.Preferably, the inhibitor of BMP is NOGGIN and the inhibitor of TGF-βsignaling is SB-431542.

In certain embodiments, the invention thus provides cell populationsenriched for anterior foregut endoderm cells. Enriched populations ofanterior foregut endoderm comprise at least 25%, at least 50%, at least75%, at least 90%, at least 95% at least 99% or at least 99.9% anteriorforegut endoderm cells.

Endoderm cell populations are characterized and distinguished by markersknown in the art. Within definitive endoderm, the embryonic stem cellmarker SOX2 reemerges as a marker of anterior foregut endoderm, whileCDX2 is a marker of posterior endoderm (hindgut). Prolonged culture ofcells induced for 4 to 5 days to form endoderm by Activin A leads to anincrease of CDX2 and a loss of SOX2, suggesting posteriorization inthese conditions. Anteriorization of definitive endoderm may beaccomplished by withdrawing or blocking Activin A and addinganteriorizing morphogens. Preferred anteriorizing morphogens areinhibitors of BMP and TGF-β signaling. Inhibitors of BMP and TGF-βsignaling may be used singly or in combination. Preferably, inhibitorsof BMP and TGF-β signaling are used in combination. Examples of BMPinhibitors are Noggin, Chordin, and follistatin. A preferred inhibitorof BMP is Noggin. Examples of inhibitors of TGF-β signaling are Ly364947(SD208), SM16, SB-505124, SB-431542, and anti-TGF-β antibodies. Apreferred inhibitor of TGF-β signaling is SB-431542. In a preferredembodiment, a combination of Noggin and SB-431542 is used to induceanteriorization of definitive endoderm. In certain embodiments, acombination of Noggin and SB-431542 is added for about 2 days in cultureto induce anteriorization of definitive endoderm. Anteriorization ofdefinitive endoderm with Noggin and SB-431542 may be confirmed by, forexample, detecting expression of, for example, one or more of SOX2, TBX1(pharynx), PAX9 (pharynx, thymus), FOXP2 (lung, airway epithelium), DLX3(esophagus), FOXA2 (definitive endoderm), and/or SOX7 (early endodermalmarker); and optionally detecting lack of expression of PAX6 (ectoderm)and/or BRACHYURY (mesoderm).

In certain embodiments, the invention provides a method of derivinganterior foregut endoderm comprising culturing definitive endoderm withan inhibitor of BMP or an inhibitor of TGF-β signaling and in theabsence of Activin A. In preferred embodiments, definitive endoderm iscultured with both an inhibitor of BMP and an inhibitor of TGF-βsignaling and in the absence of Activin A.

In some embodiments, an inhibitor of BMP is Noggin. In some embodimentsNoggin is present in cultures at a concentration of about 1 ng/ml to 10μg/ml, 10 ng/ml to 1 μg/ml, 10 ng/ml to 500 ng/ml, 10 ng/ml to 250ng/ml, or 10 ng/ml to 100 ng/ml. In preferred embodiments, Noggin ispresent in cultures at a concentration of about 25 ng/ml to 150 ng/ml,50 ng/ml to 150 ng/ml or 75 ng/ml to 150 ng/ml. In most preferredembodiments, Noggin is present in cultures at a concentration of about100 ng/ml.

In some embodiments, an inhibitor of TGF-β is SB-431542. In someembodiments of the methods described herein, SB-431542 is present incultures at a concentration of about 0.1 μM to 100 mM, 1 μM to 10 mM, 1μM to 500 μM, 1 μM to 250 μM, or 1 μM to 100 μM. In most preferredembodiments, SB-431542 is present in cultures at a concentration ofabout 10 mM.

Also preferred are embodiments wherein cultures used in the methods ofthe invention comprise Noggin at a concentration of about 75 ng/ml to150 ng/ml and SB-431542 at a concentration of about 500 μM to 10 mM.Preferably, Noggin is at a concentration of about 200 ng/ml andSB-431542 is at a concentration of about 10 mM.

With respect to ECM proteins, the definitive endoderm cell populationcan be cultured in the presence of ECM proteins. That is, the presentinvention is based on the discovery that culturing definitive endodermcells in the presence of the combination of ECM proteins and culturemedium supplemented with NOGGIN and SB-431542 resulted in highlyefficient anterior forgut endoderm differentiation. In one embodiment,the ECM proteins useful for anterior forgut endoderm differentiationmimic ECM proteins that exist during embryogenesis for lung development(e.g., collagens, laminin, fibronectin, tenascin, elastin, and a numberof proteoglycans and glycosaminoglycans).

In another embodiment, the present invention provides a method ofculturing cells in the presence of ECM proteins on a surface (e.g.,two-dimensional or three-dimensional) in a suitable growth medium. Inone embodiment, the ECM is coated on the surface of a culturingapparatus.

In another embodiment, the present invention includes a tissue culturesystem. In various aspects, the culture system is composed of the ECMcompositions described herein, such as being included in two-dimensionalor three-dimensional support materials. In another aspect, the ECMcompositions described herein serve as a support or two-dimensional orthree-dimensional support for the growth of various cell types. Forexample, the culture system can be used to support the growth of stemcells. In one aspect, the culture system can be used to support thedifferentiation of stem cells. In yet another embodiment, the culturesystem can be used to support the differentiation of definitive endodermcells into cells of the anterior foregut endoderm.

ECM is known to be secreted by certain cells and is comprised mainly offibrous proteins, polysaccharides, and other minor constituents. Itscomponents include structural elements such as collagen and elastin,adhesive proteins such as the glycoproteins fibronectin, laminin,vitronectin, thrombospondin I and tenascins, as well as proteoglycanssuch as decorin, biglycan, chondroitin sulfate and heparin sulfate andglycosaminoglycans (GAG) such as hyaluronic acid (HA).

In one embodiment, the ECM compositions can include any or all of thefollowing: fibronectin, fibrillin, laminin, elastin, members of thecollagen family (e.g., collagen I, III, and IV), glycosaminoglycans,ground substance, reticular fibers and thrombospondin. Preferably, humanECM is used for culturing the definitive endoderm. In one embodiment,human ECM includes collagens, laminin, fibronectin, tenascin, elastin,and a number of proteoglycans and glycosaminoglycans.

In another embodiment, it is desirable to culture the cells on a solidsupport that comprises a reconstituted basement membrane, wherein themembrane can be obtained by being extracted and prepared from a suitablecell tissue that is contained in the thin, membranous extracellularmatrix present below the cell layer in vivo and contains proteins andglycoproteins such as laminin, collagen IV and heparin sulphateproteoglycan as well as various cell growth factors and activatingfactors, etc.

In one embodiment, the predominant major extracellular matrix componentis fibrillar collagen, particularly collagen type I. However, otherfibrillar and non-fibrillar collagens, including collagen types II, III,IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII,XIX, and others.

The ECM compositions of the present invention may be processed in avariety of ways. Accordingly, in one embodiment, the present inventionincludes a tissue culture system. In various aspects, the culture systemis composed of the ECM compositions described herein. The ECMcompositions of the present invention may be incorporated into thetissue culture system in a variety of ways. For example, compositionsmay be incorporated as coatings, by impregnating three-dimensionalscaffold materials as described herein, or as additives to media forculturing cells. Accordingly, in one aspect, the culture system caninclude three-dimensional support materials impregnated with any of theECM compositions described herein, such as growth factors or embryonicproteins.

Alveolar Type II Progenitor Cells

Cells that are able to differentiate into the alveolar epithelial systemare cells involved in tissue repair after a lung injury, and aretherefore clinically very important cells for regenerative medicine,etc. Furthermore, these cells are also useful as materials fordiscovering new markers for identifying human lung tissue stem cells,and it is believed that analyzing the differentiation signals, etc. ofthese cells may lead to the discovery of new drugs.

In one embodiment, the anterior foregut endoderm cells of the inventioncan be induced to differentiate into a desired cell type by culturingthe cells in an appropriate differentiation medium.

Differentiation can be induced using one or more differentiation agents,including without limitation, Ca²⁺, an epidermal growth factor (EGF), aplatelet derived growth factor (PDGF), a keratinocyte growth factor(KGF), a transforming growth factor (TGF), cytokines such as aninterleukin, an interferon, or tumor necrosis factor, retinoic acid,transferrin, hormones (e.g., androgen, estrogen, insulin, prolactin,triiodothyronine, hydrocortisone, or dexamethasone), sodium butyrate,TPA, DMSO, NMF (N-methyl formamide), DMF (dimethylformamide), or matrixelements such as collagen, laminin, heparan sulfate).

In one embodiment, the anterior foregut endoderm cells of the inventioncan be induced to differentiate into cells having a lung phenotype. Forexample, anterior foregut endoderm cells can be induced to differentiateinto type II alveolar cells, which also are known as type IIpneumocytes. A medium can be used that contains one or more of pituitaryextract (e.g. a bovine pituitary extract), steroid hormones (e.g.hydrocortisone, or a salt thereof such as the acetate), growth factors(e.g., epidermal growth factor, preferably human epidermal growthfactor), catecholamines (e.g., epinephrine, either in racemic orenantiomeric form), iron-binding proteins (e.g., a transferrin),insulin, vitamins (e.g., retinoic acid), thyroid hormones (e.g.,triiodothyronine), serum albumins (e.g., bovine or human serum albumin,including recombinant preparations), antibiotics (e.g., aminoglycosideantibiotics, such as gentamicin), and/or antifingals (e.g.,amphotericin-B). For example, a medium can include hydrocortisone,epidermal growth factor, insulin, triiodothyronine, transferrin, andbovine serum albumin and in some embodiments, further can includeretinoic acid, pituitary extract, and epinephrine. SAGM™ medium fromCambrex (catalog CC-3118) is particularly useful for differentiatinganterior foregut endoderm cells into type II alveolar cells.

The present inventors have been able to obtain, through use ofappropriate differentiation factors, a pure population of lung cells.Preferably, the lung cell is a distal lung cell type, preferably analveolar-type cell, more preferably, a type-I or type-II alveolar-typecell.

In some embodiments, the methods of the invention efficiently inducedirect differentiation of stem cells into alveolar type II cells. Insome embodiments, the method results in a sufficiently pure populationof alveolar type II cells (e.g., at least 95% alveolar type IIphenotype).

In one embodiment, the anterior foregut endoderm cells of the inventioncan be induced to differentiate into cells that exhibit at least onecharacteristic of an alveolar type II cell. For example, the anteriorforegut endoderm cells can be cultured in the presence of adifferentiation medium comprising FGF-10, EGF, Wnt3a, and KGF for aperiod of time sufficient for differentiation towards an alveolar typeII cell phenotype

In some embodiments, an agonist of Wnt signaling is Wnt3a; others canalso be used, as described herein. For use in the methods describedherein, Wnt3a is present in cultures at a concentration of about 1 ng/mlto 10 μg/ml, 10 ng/ml to 1 μg/ml, 10 ng/ml to 500 ng/ml, 10 ng/ml to 250ng/ml, or 10 ng/ml to 100 ng/ml. In preferred embodiments, Wnt3a ispresent in cultures at a concentration of about 25 ng/ml to 150 ng/ml,50 ng/ml to 150 ng/ml or 75 ng/ml to 150 ng/ml. In further preferredembodiments, Wnt3a is present in cultures at a concentration of about100 ng/ml.

In some embodiments, agonists of FGF signaling are FGF7 or FGF10; otherscan also be used, as described herein. For use in the methods describedherein, FGF7 or FGF10 are present in cultures at a concentration ofabout 1 ng/ml to 10 μg/ml, 10 ng/ml to 1 μg/ml, 10 ng/ml to 500 ng/ml,10 ng/ml to 250 ng/ml, or 10 ng/ml to 100 ng/ml. In preferredembodiments, FGF7 or FGF10 are present in cultures at a concentration ofabout 25 ng/ml to 150 ng/ml, 50 ng/ml to 150 ng/ml or 75 ng/ml to 150ng/ml. In most preferred embodiments, both FGF7 and FGF 10 are presentin cultures at a concentration of about 10 ng/ml.

Exemplary stem cell differentiated alveolar type II cells appearmorphologically normal, express the characteristic surfactant proteinsA, B, and C, CFTR and α-1AT RNA as well as synthesize and secretecomplement proteins C3 and C5. Thus, a unique approach is provided toreliably generate significant quantities of sufficiently pure stemcell-derived alveolar type II cells that can be used therapeutically toreconstitute damaged lung alveolus and other lung diseases or disorderssuch as, but not limited to, genetic diseases that affect the lung.

Preferably, the cells form histiotypic alveolar-like structures,comprised of differentiated distal epithelial cells (proSpC expressing)forming ductal structures. Thus, the implanted cells will developcharacteristics that liken it to the surrounding tissue. Using thesemethods, the biological scaffolding can augment the tissue; thebiological scaffolding of the invention can be used for tissueengineering and in any conventional tissue engineering setting.

In one embodiment, the present methods result in direct differentiationof stem cells into alveolar type II cells which contrasts with previousattempts at differentiation of alveolar type II cells from stem cells,in which multiple steps were used to derive alveolar type II cells fromstem cells through embryonic body formation. Previous approaches requireprolonged time periods to develop the endoderm from which the alveolartype II cells are derived, and yet in the end the produce scarcelydetectable numbers of alveolar type II cells in mixed cell populations.Therefore, in addition to providing sufficiently pure and numerousalveolar type II cells, embodiments of the present methods decrease thetime and effort in generating stem cell-derived alveolar type II cellsand facilitate their therapeutic and clinical use.

Differentiation to lung cells (e.g., alveolar type II cells) can beconfirmed, for example, by a lung morphology as assessed by lightmicroscopy and the presence of lamellar bodies and microvesicular bodiesas assessed by transmission electron microscopy. Lamellar bodies aresecretory lysosomes that serve as the storage form of lung surfactant,surfactant protein C (SPC), which is an integral membrane protein thatis expressed only in alveolar type II cells. The presence of SPC mRNAcan be detected by reverse-transcriptase PCR and the presence of SPCprotein can be detected by immunofluorescence staining.

Methods

The invention relates to the discovery that stem cells (e.g., iPS cells)can be differentiated to alveolar type II cells by way of the definitiveendoderm. For example in one embodiment, the invention provides a methodof differentiating a population of stem cells into a population of lungcells comprising: 1) inducing a stem cell into a cell of the definitiveendoderm; 2) inducing the cell of the definitive endoderm into ananterior foregut endoderm cell; 3) inducing the anterior foregutendoderm cell into a lung cell, thereby differentiating a stem cell intoa lung cell. The cells of the invention are useful for investigatinglung developmental biology. In addition, the cells of the invention areuseful for among other things, drug discovery, toxicity testing, diseasepathology, and the like. Accordingly, the invention provides methods andcompositions for the generation of vascularized pulmonary tissues as aform of regenerative medicine.

The production of a population of in vitro cultured cells of alveolarepithelial type II cell lineage derived from at least one stem cellincludes culturing at least stem cell in vitro according to the methodof the invention in order to produce differentiated cells, preferablywithout formation of an embryonic body. In one embodiment, the method ofproduction further includes identifying the differentiated cells ofalveolar epithelial type II cell phenotype by detecting expression of atleast one biomarker of alveolar epithelial type II cells, and isolatingthe differentiated cells having alveolar epithelial type II cellphenotype. In some cases, this may include selecting a purifiedpopulation of differentiated cells wherein at least 95%, preferably atleast 96%, preferably at least 97%, more preferably at least 98%, morepreferably at least 99% of the cells have alveolar epithelial type IIcell phenotype.

The cells of the invention and cells derived therefrom can be derivedfrom, inter alia, humans, primates, rodents and birds. Preferably, thecells of the invention are derived from mammals, especially mice, ratsand humans. Stem cells from which the of alveolar epithelial type IIcells are derived may be either wild-type or genetically modified stemcells.

The cells of the present invention, whether grown in suspension or asadherent cell cultures, are grown in contact with culture media.

Culture media used in the present invention preferably comprise a basalmedium, optionally supplemented with additional components.

Basal medium is a medium that supplies essential sources of carbonand/or vitamins and/or minerals for the cells. The basal medium isgenerally free of protein and incapable on its own of supportingself-renewal/symmetrical division of the cells.

Preferably, the suitable cell is isolated from a mammal, more preferablya primate and more preferably still, a human. The cells useful in themethods of the present invention are isolated using methods discussedherein, for example in the Examples section, or by any method known inthe art. Following isolation, the suitable cells are cultured in aculture medium. Media formulations that support the growth of cellsinclude, but are not limited to, Minimum Essential Medium Eagle, ADC-1,LPM (bovine serum albumin-free), F10 (HAM), F12 (HAM), DCCM1, DCCM2,RPMI 1640, BGJ Medium (with and without Fitton-Jackson Modification),Basal Medium Eagle (BME—with the addition of Earle's salt base),Dulbecco's Modified Eagle Medium (DMEM-without serum), Yamane, IMEM-20,Glasgow Modification Eagle Medium (GMEM), Leibovitz L-15 Medium, McCoy's5A Medium, Medium M199 (M199E—with Earle's salt base), Medium M199(M199H—with Hank's salt base), Minimum Essential Medium Eagle(MEM-E—with Earle's salt base), Minimum Essential Medium Eagle(MEM-H—with Hank's salt base) and Minimum Essential Medium Eagle(MEM-NAA with nonessential amino acids), and the like.

It is further recognized that additional components may be added to theculture medium. Such components include, but are not limited to,antibiotics, antimycotics, albumin, growth factors, amino acids, andother components known to the art for the culture of cells. Antibioticswhich can be added into the medium include, but are not limited to,penicillin and streptomycin. The concentration of penicillin in theculture medium is about 10 to about 200 units per ml. The concentrationof streptomycin in the culture medium is about 10 to about 200 μg/ml.However, the invention should in no way be construed to be limited toany one medium for culturing the cells of the invention. Rather, anymedia capable of supporting the cells of the invention in tissue culturemay be used.

In certain embodiments, culture media used in the invention do notcontain any components which are undefined (e.g. serum and/or feedercells), that is to say components whose content is unknown or which maycontain undefined or varying factors that are unspecified. An advantageof using fully defined media, free of serum and free of serum extracts,is that efficient and consistent protocols for culture and subsequentmanipulation of the cells of the invention and cells derived therefromcan be obtained.

Typical substrates for culture of the cells in all aspects of theinvention are culture surfaces recognized in this field as useful forcell culture, and these include surfaces of plastics, metal, composites,though commonly a surface such as a plastic tissue culture plate, widelycommercially available, is used. Such plates are often a few centimetersin diameter. For scale up, this type of plate can be used at much largerdiameters and many repeat plate units used.

The culture surface may further comprise a cell adhesion protein,usually coated onto the surface. Receptors or other molecules present onthe cells bind to the protein or other cell culture substrate and thispromotes adhesion to the surface and promotes growth.

In certain embodiments, the cultures of the invention are preferablyadherent cultures, i.e. the cells are attached to a substrate.

In some instances, the cells of the invention can be cultured on adecellularized tissue, preferably a decellularized lung tissue. In someinstances, cells of the invention are cultured on the decellularizedtissue for regeneration of lung tissue. An example of a decellularizedlung tissue is disclosed in PCT/US2010/023213, the content of which isincorporated herein by reference in its entirety.

In one embodiment, the invention provides cells that “seed” thedecellularized tissue scaffold. The cells can differentiate in vitro byculturing the cells on the scaffold in the presence of an appropriatedifferentiation medium. Differentiated cells can be identified by theirgross morphology and by the connections they form with other cells. Forexample, cells that differentiate into lung cells can develop complexmorphology resembling bronchioles.

The present invention also provides an in vivo method of repairinginjured or diseased alveolar epithelial tissue in the lung of a mammalis provided in accordance with some embodiments. Such method comprisestransplanting into a lung that contains injured or diseased alveolarepithelial tissue, a population of differentiated stem cells, or progenythereof, at least 95% of which have alveolar epithelial type IIphenotype. The population of cells is prepared in accordance with amethod described herein, and is effective to repair at least a portionof the injured or diseased alveolar epithelial tissue. In someembodiments, the mammal suffers from a genetic disease affectingalveolar epithelial tissue in the lung, and said therapeutic transgeneencodes a gene product for ameliorating the detrimental effects of saidgenetic disease in said alveolar epithelial tissue. In some embodiments,at least one differentiated stem cell, or progeny thereof, comprises atherapeutic transgene operably linked to a cell-specific promoter,wherein the transgene encodes a therapeutic gene product. In someembodiments, an above-described population of cells is transplanteddirectly to injured or diseased alveolar epithelial tissue in said lung.In some embodiments, transplanting the population of cells comprisesadministering them into the lung endotracheally via oropharynxintubation.

In one embodiment, the methods of the invention provides the generationof lung progenitor cells from differentiated cultures of stem cells.Moreover, with the recent discovery that iPS cells can be created fromdermal skin fibroblasts, investigations have begun to examine thepossibility of generating lung progenitor epithelial cells from somaticcells. If these efforts succeed, patient-specific iPS cells could beobtained, thereby avoiding the immune rejection problems that mightoccur if heterologous sources of embryonic stem cells were employed.Moreover, iPS cells offer the possibility of generating gene-corrected,patient-specific lung progenitor cells from individuals with geneticdiseases affecting the lung, including cystic fibrosis,alpha-1-antitrypsin deficiency, and surfactant protein deficiencies.

Many embodiments of the present compositions and methods efficientlyinduce direct differentiation of human stem cells into alveolarepithelial type II cells without EB formation and also produce a highlypure population of human alveolar epithelial type II cells. In someembodiments the method results in a clonal population of alveolarepithelial type II phenotype cells sufficiently pure (e.g., at least 95%and in many cases at least 99% alveolar epithelial type II phenotype)suitable for implantation into a mammalian host lung tissue withoutsignificant risk of producing a teratoma.

Transplantation of the stem cell-derived alveolar epithelial type IIcells, produced using embodiments of the described methods reversed orprevented acute lung injury when transplanted 1 or 2 days followinginjury, as demonstrated by recovery of body weight and arterial bloodoxygen saturation, decreased collagen deposition, and increasedsurvival. Interestingly, some injured lung alveolar epithelium regionsappeared healthy after transplantation of the stem cell-derived alveolarepithelial type II cells, produced using embodiments of the describedmethods, despite having no observable engrafted stem cell-derivedalveolar epithelial type II cells. This suggests that stem cell-derivedalveolar epithelial type II cells, produced using embodiments of thedescribed methods, may provide paracrine repair/protection to injuredlung epithelium, such as, but not limited to, the release ofanti-inflammatory mediators such as IL-10, angiopoietin-1, andkeratinocyte growth factor.

In some embodiments, the alveolar type II epithelial cells derived fromstem cells (such as but not limited to human iPS cells, as used in theexamples) is used therapeutically in the treatment of lung injury.Therapeutic activity is demonstrated herein using alveolar type IIepithelial cells derived from stem cells in an animal model of acutelung injury. When transplanted into lungs of mice subjected tobleomycin-induced acute lung injury, stem cell derived-alveolarepithelial type II cells behaved as normal primary alveolar epithelialtype II cells, differentiating into cells expressing phenotypic markersof alveolar type I epithelial cells. Without experiencing tumorigenicside effects, lung injury was abrogated in mice transplanted with stemcell derived-alveolar epithelial type II cells, demonstrated by recoveryof body weight and arterial blood oxygen saturation, decreased collagendeposition, and increased survival. Therefore, transplantation of stemcell derived-alveolar epithelial type II cells shows promise as aneffective therapeutic to treat acute lung injury and related disorders.

In one embodiment, the invention provides a method of repairing injuredor diseased alveolar epithelial tissue in the lung of a mammalcomprising transplanting a population of differentiated stem cells, orprogeny thereof of the invention into the mammal. Preferably, cells ofthe invention are transplanted at a site comprising injured or diseasedalveolar epithelial tissue. In another embodiment, the cells of theinvention are transplanted directly into the lung of the mammal. In oneembodiment, the population of differentiated stem cells, or progenythereof of the invention is at least 95%, preferably at least 96%,preferably at least 97%, more preferably at least 98%, more preferablyat least 99% of which exhibit alveolar epithelial type II phenotype,wherein the population of cells is prepared in accordance with themethods of the invention, and is effective to repair at least a portionof the injured or diseased alveolar epithelial tissue at the site. Thedifferentiated stem cell, or progeny thereof, may comprise a transgene,which encodes a desirable gene product (e.g., a therapeutic protein orpeptide), operably linked to a cell-specific promoter.

In one embodiment, the invention provides a method of treating a geneticdisease affecting alveolar epithelial tissue in the lung of a mammalcomprising transplanting a population of differentiated stem cells, orprogeny thereof of the invention into the mammal. Preferably, the cellsof the invention are transplanted into the lung, at a site comprisingalveolar epithelial tissue detrimentally affected by the geneticdisease. The differentiated stem cell, or progeny thereof of theinvention can comprise a transgene that encodes a gene product whichameliorates the genetic disease or its detrimental effects in thealveolar epithelial tissue at least at the site of implantation whenexpressed in vivo. The cells of the invention may comprise a transgeneoperably linked to a cell-specific promoter, wherein the transgeneencodes a therapeutic gene product.

Methods of treatment of the diseases encompassed by the invention cancomprise the transplantation of single cells, cell lines, compositions,or cell populations of the invention into a mammal in need thereof.Preferably, the mammal is a human.

In one embodiment, the cells of the invention can be used to assay theeffectiveness of inductive or blocking factors on the differentiation ofthe cells of the invention. Such an assay may comprise contacting a cellof the invention (i.e. as present in the compositions, cell lines, andpopulations, or a single cell) with the factor to be tested. The effectof the factor on the differentiation of the cell can be suitablyassessed by determining the marker profile of the resultant cells, i.e.to show whether the cells have a similar marker profile to the cells ofthe invention, or whether these markers have been lost. The cells of theinvention are also suitable for assaying pharmaceuticals, for example,the treatment of lung disease.

Genetic Modification

The cells of the invention can be used to treat a lung disease includingbut not limited to emphysema, bronchiolitis obliterans, and cysticfibrosis. For example, cells and be delivered to a recipient viatracheal instillation, inhalation, or injection, among other ways. Suchcells that are expanded in culture can be used to affect therapy in therecipient.

In the context of gene therapy, the cells of the invention can betreated with a gene of interest prior to delivery of the cells into thelung of a recipient. In some cases, such cell-based gene delivery canpresent significant advantages of other means of gene delivery to thelung, such as inhalation of adenoviral gene delivery vectors. Thissuperiority of cell-based gene delivery to a host stems from theobservation that inhaled gene delivery vectors typically result in poorefficiency of cellular transduction, due to barriers imposed by themucous layer and the host immune system. Delivery of a therapeutic genethat has been pre-inserted into cells avoids the problems associatedwith penetration of gene therapy vectors into recipient lung cells.

Accordingly, the invention provides the use of genetically modifiedcells that have been cultured according to the methods of the invention.Genetic modification may, for instance, result in the expression ofexogenous genes (“transgenes”) or in a change of expression of anendogenous gene. Such genetic modification may have therapeutic benefit.Alternatively, the genetic modification may provide a means to track oridentify the cells so-modified, for instance, after implantation of acomposition of the invention into an individual. Tracking a cell mayinclude tracking migration, assimilation and survival of a transplantedgenetically-modified cell. Genetic modification may also include atleast a second gene. A second gene may encode, for instance, aselectable antibiotic-resistance gene or another selectable marker.

Proteins useful for tracking a cell include, but are not limited to,green fluorescent protein (GFP), any of the other fluorescent proteins(e.g., enhanced green, cyan, yellow, blue and red fluorescent proteins;Clontech, Palo Alto, Calif.), or other tag proteins (e.g., LacZ,FLAG-tag, Myc, His₆, and the like).

When the purpose of genetic modification of the cell is for theproduction of a biologically active substance, the substance willgenerally be one that is useful for the treatment of a given disorder.For example, it may be desired to genetically modify cells so that theysecrete a certain growth factor product associated with bone or softtissue formation. Growth factor products to induce growth of other,endogenous cell types relevant to tissue repair are also useful. Forinstance, growth factors to stimulate endogenous capillary and/ormicrovascular endothelial cells can be useful in repair of soft tissuedefect, especially for larger volume defects.

The cells of the present invention can be genetically modified by havingexogenous genetic material introduced into the cells, to produce amolecule such as a trophic factor, a growth factor, a cytokine, and thelike, which is beneficial to culturing the cells. In addition, by havingthe cells genetically modified to produce such a molecule, the cell canprovide an additional therapeutic effect to the mammal when transplantedinto a mammal in need thereof. For example, the genetically modifiedcell can secrete a molecule that is beneficial to cells neighboring thetransplant site in the mammal.

The cells of the invention may be genetically modified using any methodknown to the skilled artisan. See, for instance, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.), and in Ausubel et al., Eds, (1997,Current Protocols in Molecular Biology, John Wiley & Sons, New York,N.Y.). For example, a cell may be exposed to an expression vectorcomprising a nucleic acid including a transgene, such that the nucleicacid is introduced into the cell under conditions appropriate for thetransgene to be expressed within the cell. The transgene generally is anexpression cassette, including a polynucleotide operably linked to asuitable promoter. The polynucleotide can encode a protein, or it canencode biologically active RNA (e.g., antisense RNA or a ribozyme).Thus, for example, the polynucleotide can encode a gene conferringresistance to a toxin, a hormone (such as peptide growth hormones,hormone releasing factors, sex hormones, adrenocorticotrophic hormones,cytokines (e.g., interferins, interleukins, lymphokines, etc.), acell-surface-bound intracellular signaling moiety (e.g., cell adhesionmolecules, hormone receptors, etc.), a factor promoting a given lineageof differentiation (e.g., bone morphogenic protein (BMP)), etc.

Within the expression cassette, the coding polynucleotide is operablylinked to a suitable promoter. Examples of suitable promoters includeprokaryotic promoters and viral promoters (e.g., retroviral ITRs, LTRs,immediate early viral promoters (IEp), such as herpesvirus IEp (e.g.,ICP4-IEp and ICPO-IEEp), cytomegalovirus (CMV) IEp, and other viralpromoters, such as Rous Sarcoma Virus (RSV) promoters, and MurineLeukemia Virus (MLV) promoters). Other suitable promoters are eukaryoticpromoters, such as enhancers (e.g., the rabbit .beta.-globin regulatoryelements), constitutively active promoters (e.g., the .beta.-actinpromoter, etc.), signal specific promoters (e.g., inducible promoterssuch as a promoter responsive to RU486, etc.), and tissue-specificpromoters. It is well within the skill of the art to select a promotersuitable for driving gene expression in a predefined cellular context.The expression cassette can include more than one coding polynucleotide,and it can include other elements (e.g., polyadenylation sequences,sequences encoding a membrane-insertion signal or a secretion leader,ribosome entry sequences, transcriptional regulatory elements (e.g.,enhancers, silencers, etc.), and the like), as desired.

The expression cassette containing the transgene should be incorporatedinto a genetic vector suitable for delivering the transgene to thecells. Depending on the desired end application, any such vector can beso employed to genetically modify the cells (e.g., plasmids, naked DNA,viruses such as adenovirus, adeno-associated virus, herpesviruses,lentiviruses, papillomaviruses, retroviruses, etc.). Any method ofconstructing the desired expression cassette within such vectors can beemployed, many of which are well known in the art (e.g., direct cloning,homologous recombination, etc.). The choice of vector will largelydetermine the method used to introduce the vector into the cells (e.g.,by protoplast fusion, calcium-phosphate precipitation, gene gun,electroporation, DEAE dextran or lipid carrier mediated transfection,infection with viral vectors, etc.), which are generally known in theart.

Examples of techniques sufficient to direct persons of skill through invitro amplification methods, including the polymerase chain reaction(PCR), the ligase chain reaction (LCR), and other DNA or RNApolymerase-mediated techniques are found in Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL, volumes 1-3 (3^(rd) ed., Cold SpringHarbor Press, N Y 2001).

Once the nucleic acid for a protein is cloned, a skilled artisan mayexpress the recombinant gene(s) in a variety of lung cells. It isexpected that those of skill in the art are knowledgeable in thenumerous expression systems available for expressing the desiredtransgene.

Bioreactor

The invention provides a system (e.g., a bioreactor) for culturing thecells of the invention that have been introduced to a decellularizedlung. The bioreactor enables the maintenance of cell viability, cellulardifferentiation state, and lung morphology. The bioreactor of theinvention incorporates key features of the vivo environment. Thebioreactor can be designed to allow modifications for optimizingdecellularization and/or recellularization processes.

In one embodiment, the bioreactor is capable of perfusing media throughthe vasculature at a rate specified by the user and within thephysiological flow and pressure levels of a mammal. In anotherembodiment, the bioreactor is capable of ventilating the tissue (e.g.,lung) with air or media through the trachea. Preferably, negativepressure ventilation is used in order to be consistent with normalphysiological conditions, though ventilation using positive pressure canalso be done. In yet another embodiment, the bioreactor is capable ofallowing different media types to bathe the vascular and airwaycompartments of the tissue. In another embodiment, the bioreactor allowsfor gas exchange into the culture medium, while simultaneously meetingthe desired requirements for ventilation. In another embodiment, thebioreactor has ports to allow for pressure measurements, for examplemeasurements of the pulmonary artery and tracheal pressures. Preferably,pressures are within normal physiological values. In another embodiment,the bioreactor has a means of allowing media exchange on a periodicbasis.

The bioreactor of the invention generally includes at least onecannulation device for cannulating a tissue, a perfusion apparatus forperfusing media through the cannula(s), and means (e.g., a containmentsystem) to maintain a sterile environment for the organ or tissue. Acannulation device generally includes size-appropriate hollow tubing forintroducing into a vessel, duct, and/or cavity of a tissue. Typically,one or more vessels, ducts, and/or cavities are cannulated in a tissue.A perfusion apparatus can include a holding container for the liquid(e.g., a cellular disruption medium) and a mechanism for moving theliquid through the organ (e.g., a pump, air pressure, gravity) via theone or more cannulae. The sterility of a tissue during decellularizationand/or recellularization can be maintained using the methods discussedelsewhere herein.

The bioreactor for can be used to recellularize tissues as describedherein. The process can be monitored for certain perfusioncharacteristics (e.g., pressure, volume, flow pattern, temperature,gases, pH), mechanical forces (e.g., ventricular wall motion andstress), and electrical stimulation (e.g., pacing). The effectiveness ofperfusion can be evaluated in the effluent and in tissue sections.Perfusion volume, flow pattern, temperature, partial O₂ and CO₂pressures and pH can be monitored using standard methods.

Sensors can be used to monitor the bioreactor and/or the tissue.Sonomicromentry, micromanometry, and/or conductance measurements can beused to acquire pressure-volume. For example, sensors can be used tomonitor the pressure of a liquid moving through a cannulated organ ortissue; the ambient temperature in the system and/or the temperature ofthe organ or tissue; the pH and/or the rate of flow of a liquid movingthrough the cannulated organ or tissue; and/or the biological activityof a recellularizing tissue. In addition to having sensors formonitoring such features, a system for recellularizing a tissue also caninclude means for maintaining or adjusting such features. Means formaintaining or adjusting such features can include components such as athermometer, a thermostat, electrodes, pressure sensors, overflowvalves, valves for changing the rate of flow of a liquid, valves foropening and closing fluid connections to solutions used for changing thepH of a solution, a balloon, an external pacemaker, and/or a compliancechamber. To help ensure stable conditions (e.g., temperature), thechambers, reservoirs and tubings can be water-jacketed.

The bioreactor is capable of providing sufficient nutrient supply andmechanical stimulation to the lung tissue in order to support cellsurvival and differentiation. The bioreactor can be used for in vitrolung tissue culture and for engineered lung tissue culture. Preferably,the bioreactor is used to culture engineered lung tissue using thedecellularized lung scaffolds in combination with the cells of theinvention.

The development of a bioreactor capable of the in vitro culture of true3-dimensional segments of lung tissue is an important step in thedevelopment of clinically useful engineered lung tissue. For example,growth and maturation of the engineered lung tissue can take place inthe bioreactor prior to implantation of the engineered lung into arecipient, thereby enhancing the functionality of the final implantedlung tissue in vivo. In addition, the bioreactor for in vitro lungculture can be used to assist the study of pulmonary biology,physiology, and development. That is, the interactions of lungendothelial and epithelial cells to form the alveolar-capillary barriercan be studied using the engineered lung tissue and bioreactor of theinvention. A skilled artisan would be able to study lung behavior in amore controlled environment than the various animal models currentlyused. The engineered lung tissue and bioreactor could also be used forpharmacologic testing and investigation in human or animal tissue beforeproceeding to time-consuming and costly human or animal trials.

Administration

The invention contemplates use of the cells of the invention in both invitro and in vivo settings. Thus, the invention provides for use of thecells of the invention for research purposes and for therapeutic ormedical/veterinary purposes. In research settings, an enormous number ofpractical applications exist for the technology. One example of suchapplications is use of the cells of the invention in an ex vivo cancermodel, such as one to test the effectiveness of various ablationtechniques (including, for example, radiation treatment, chemotherapytreatment, or a combination) in a lab, thus avoiding use of ill patientsto optimize a treatment method. For example, one can attach a recentlyremoved lung to a bioreactor and treat the lung to ablate tissue.Another example of an in vivo use is for tissue engineering.

The invention also provides a method of alleviating or treating a lungdefect in a mammal, preferably a human. The method comprisesadministering to the mammal in need thereof a therapeutically effectiveamount of a composition comprising the cells of the invention, therebyalleviating or treating the lung defect in the mammal.

The cells of the present invention have use in vivo. Among the varioususes, mention can be made of methods of in vivo treatment of subjects(used interchangeably herein with “patients”, and meant to encompassboth human and animals). In general for certain embodiments, methods oftreating subjects comprise implanting a cell of the invention into or onthe surface of a subject, where implanting of the cell results in adetectable change in the subject. The detectable change can be anychange that can be detected using the natural senses or using man-madedevices. While any type of treatment is envisioned by the presentinvention (e.g., therapeutic treatment of a disease or disorder,cosmetic treatment of skin blemishes, etc.), in many embodiments, thetreatment is a therapeutic treatment of a disease, disorder, or otheraffliction of a subject. As such, a detectable change may be detectionof a change, preferably an improvement, in at least one clinical symptomof a disease or disorder affecting the subject. Exemplary in vivotherapeutic methods include regeneration of organs after treatment for atumor, preparation of a surgical site for implantation of a medicaldevice, skin grafting, and replacement of part or all of a tissue ororgan, such as one damaged or destroyed by a disease or disorder. Inview of the fact that a subject may be a human or animal, the presentinvention has both medical and veterinary applications.

The invention also provides methods of treating a patient by implantingthe cells of the invention into a mammal in need thereof. In someinstances, the cells of the invention comprise suitable cells, forexample alveolar epithelial type II cells. However, the invention shouldnot be limited to any particular type of cells. After implantation, thegrafted cells can respond to environmental cues that will cause it todevelop characteristics of the endogenous tissue. Preferably, the cellsform histiotypic alveolar-like structures, comprised of differentiateddistal epithelial cells (proSpC expressing) forming ductal structures.Thus, the implanted cells will develop characteristics that liken it tothe surrounding tissue. Using these methods, the biological scaffoldingcan augment the tissue; the biological scaffolding of the invention canbe used for tissue engineering and in any conventional tissueengineering setting.

Accordingly, the invention encompasses tissue regeneration applications.The objective of the tissue regeneration therapy approach is to deliverhigh densities of repair-competent cells (or cells that can becomecompetent when influenced by the local environment) to the defect sitein a format that optimizes both initial wound mechanics and eventualneotissue production. The composition of the instant invention isparticularly useful in methods to alleviate or treat lung tissue defectsin individuals. Advantageously, the composition of the inventionprovides for improved lung tissue regeneration. Specifically, the tissueregeneration is achieved more rapidly as a result of the inventivecomposition.

Advantageously, the compositions and methods of the invention representan improvement over prior art methods. Preferably the composition foruse in treating a lung tissue defect comprises alveolar epithelial typeII cells as described elsewhere herein.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1: Differentiation and Characterization of Alveolar Type IICells from Human Induced Pluripotent Stem Cells

The experiments presented herein were designed to explore whether lungtissue can be regenerated in vitro. A relatively homogeneous populationof alveolar epithelial type II (AETII) and type I cells (AETI) wasgenerated from human iPS cells which had phenotypic properties similarto mature human alveolar type II and type I cells. Up to 97% of cellswere positive for surfactant protein C, 95% for Mucin-1, 93% forsurfactant protein B, and 89% for the epithelial marker, CD54.Additionally, exposing AETII to a Wnt/β-catenin inhibitor (e.g., IWR-1)changed the iPSC-AETII like phenotype to a predominantly AETI likephenotype. Of the cells that were AET1 cells, more than 90% of werepositive for the type I markers, T1α and caveolin-1. Acellular lungmatrices were prepared by treating whole rat or human adult lungs withdecellularization reagents, followed by seeding these matrices withalveolar cells derived from human iPS cells. Under appropriate cultureconditions, these progenitor cells adhered to and proliferated withinthe 3D lung tissue scaffold, and displayed markers of differentiatedpulmonary epithelium.

The materials and method employed in these experiments are nowdescribed.

Chemical and Reagents

Mouse embryonic fibroblasts (MEFs) (GSC-6201) were purchased from Globalstem, Matrigel (354277) were purchased from BD, Recombinant human WNT3a(5036WN) was purchased from R&D. Dispase (07923) was obtained from StemCell Technology. Keratinocyte growth factor (KGF) (PHG0094), fibroblastgrowth factor 10 (FGF-10) (PHG0204), epidermal growth factor (EGF)(PHG0311), human basic fibroblast growth factor (bFGF) (13256-029),NOGGIN (PHC1506), Activin A (PHG9014), knock out serum replacement(108280280), Superscript first strand synthesis system for RT-PCR(18080-051) were purchased from Invitrogen. Dulbecco's Modified EagleMedium: Nutrient Mixture F-12 (DMEM/F-12) (11330-032), Dulbecco'sModified Eagle Medium (DMEM) (11905-092), RPMI 1640 (11875-093), IMDM(12440-053), non-essential amino acids (1140-050), L-glutamine(25030164), sodium pyruvate (11360-070), 2-mercaptoethanol (21985-023),B27 supplement (17504-044), Trypsin (25200-056), Penicillin-streptomycin(15140-122) fetal bovine serum (FBS) were purchased from GIBCO by lifetechnology. Retinoic acid (R2625) gelatin (G1393), SB431542(S4317),human ECM protein (E0282), IWR-1(I0161), human collagen type I (C7624)and IV (C7521), human fibronectin (F0895), human ECM protein (E0282),CHAPs C3023), Benzonase (E1014), Sodium Deoxycholate (D6750), Elastase(E8140) Triton X-100 (T9284), were purchased from Sigma-Aldrich. SodiumNitroprusside Dihydrate (71778) was obtained from Fluka. DNase I(LS006333) was purchased from Worthington Biochemical Corporation. SmallAirway Growth Medium (SAGM) (CC-3119), Amphotericin B (17836R) waspurchased from Lonza. Human SPC ELISA kit (E01S0168) was obtained fromLife Science Advanced Technologiest Inc. Peracetic acid or PAA (P05020)was obtained from Pfalz & Bauer. Fetal bovine serum (FBS) (SH30071.03)was obtained from Hyclone. iQ™ SYBR Green Supermix (170-8882) wasobtained from Bio-Rad. All antibodies were used in this study are listedin Table 1.

TABLE 1 List of antibodies used in staining, flow cytometry and westernblot for various experiments Primary Antibodies Antigen Type Provider(Cat #) (lot #) Application Beta-actin Monoclonal Abcam (Cat# ab8226,Lot# GR88207-1) WB Caspase3 Rabbit Abcam (Cat# ab13847, Lot# GR62173-IHC polyclonal 2) CCSP Rabbit Millipore (Cat# 07-623, Lot# 1972321) IHCpolyclonal CCSP Goat Biovender (Cat# RD81022220, Lot# IHC polyclonalRD2412) CD54-PE Mouse BD Pharmingen (Cat# 560971, Lot# FC Monoclonal10609) CXCR4-APC Monoclonal BD Pharmingen (Cat# 560936, Lot# FC 41560)Cytokeratin-5 Rabbit Abbiotec (Cat# 251431, Lot# 11092101) IHCpolyclonal FoxA2 Goat R&D Systems (Cat# AF2400, Lot# ICC, FC polyclonalULB0311101) Muc-1 Monoclonal R&D Systems (Cat# MAB6298, Lot# ICC, FCCDYA0111031) Nanog Rabbit Abcam (Cat# ab80892, Lot# GR40243- ICCmonoclonal 14) Nkx2.1 Rabbit Abcam (Cat# ab76013, Lot# GR76790- ICC,IHC, polyclonal 2) FC Oct4 Goat Abcam (Cat# ab27985, Lot# GR56247- ICC,FC polyclonal 1) p63 Monoclonal Santa Cruz (Cat# sc-71825, Lot# H2510)IHC Pax9 Rat monoclonal Abcam (Cat# ab28538, Lot# GR53993- ICC 1) Pax9Goat Santa Cruz (Cat# sc-7746, Lot# K121) ICC polyclonal PCNA MonoclonalAbcam (Cat# ab29, Lot# GR70504-2) IHC proSPB Rabbit Millipore (Cat#ab3430, Lot# ICC, FC polyclonal NG1820771) proSPC Rabbit Millipore (Cat#ab3786, Lot# 2117989) ICC, IHC, monoclonal FC proSPC Rabbit Abcam (Cat#ab40879, Lot# GR86765- WB polyclonal 1) Sox2 Rabbit Abcam (Cat# ab97959,Lot# Unknown) ICC polyclonal Sox2-AF647 Monoclonal BD Pharmingen (Cat#562139, Lot# ICC, FC 18245) Sox17 Goat R&D Systems (Cat# AF1924, Lot#ICC, FC polyclonal KGA0411031) Sox17 Mouse Abcam (Cat# ab84990) ICCmonoclonal SPA Rabbit Millipore (Cat# ab3420, Lot# ICC polyclonalNG1888873) SPA Rabbit Santa Cruz (Cat# sc-13977, Lot# K0807) WBpolyclonal SPC Rabbit Santa Cruz (Cat# sc-13979, Lot# L1710) ICC, IHC,polyclonal FC SPC Monoclonal Life Sciences Advanced Tech.(Cat ELISA#E01S0168) SSEA4 Monoclonal Millipore (Cat# MAB4304, Lot # ICC LV1488380T1α Monoclonal Abcam (Cat# ab10288, Lot# GR47830- IHC 3) Tbx1 RabbitAbcam (Cat # ab18530) ICC polyclonal TRA-1-81 Monoclonal Millipore(Cat#MAB4381, Lot # ICC LV1512392) CD166-PE Mouse (Cat# 559263, Lot#3018832) FC Monoclonal CD104-FITC Mouse (Cat# 64233, Lot # 3039557 FCMonoclonal Detection Antbodies Type Provider(Cat #) (lot #) ApplicationAlexa Fluor ® 555 Donkey Anti- Invitrogen (Cat# A21432, Lot# ICC, IHCGoat IgG (H + L) 439379) Alexa Fluor ® 568 Donkey Anti- Invitrogen (Cat#A10037, Lot# ICC, IHC Mouse IgG 1110068) Alexa Fluor ® 488 DonkeyAnti-Rat Invitrogen (Cat# A21208, Lot# ICC IgG (H + L) 1017330) AlexaFluor ® 488 Rabbit Anti-Goat Invitrogen (Cat# A11078, Lot# ICC, IHC IgG(H + L) 1069847) Alexa Fluor ® 555 Goat Anti-Rabbit Invitrogen (Cat#A21429, Lot# ICC, IHC IgG (H + L), highly cross-absorbed 1010124) AlexaFluor ® 488 Goat Anti-Rabbit Invitrogen (Cat# A11034, Lot# ICC, IHC IgG(H + L), highly cross-adsorbed 1008720) Alexa Fluor ® 555 GoatAnti-Mouse Invitrogen (Cat# A21424, Lot# ICC, IHC IgG (H + L), highlycross-adsorbed 1214852) Alexa Fluor ® 555 Goat Anti-Rat Invitrogen (Cat#A21434, Lot# ICC IgG (H + L) 1008806) Alexa Fluor ® 488 Chicken Anti-Invitrogen (Cat# A21441, Lot# ICC, IHC Rabbit IgG (H + L) 1003212) AlexaFluor ® 488 Chicken Anti- Invitrogen (Cat# A21467, Lot# ICC, IHC GoatIgG (H + L) 474697) Goat anti-Mouse IgG - H&L (FITC) Abcam (Cat# ab6785,Lot# ICC, IHC GR6891-4) Goat anti-Rabbit IgG-HRP Santa Cruz (Cat#sc-2004, Lot# WB H2806) HRP-conjugated Life Sciences Advanced Tech.(CatELISA #E01S0168 APC Mouse IgG2a, κ Isotype BD Pharmingen (Cat# 555576,Lot# Isotype Control 33828) control PE Mouse IgG1, κ Isotype ControleBiosciences (Cat# 12-4714-82, Isotype Lot# E01672-1630) control AlexaFluor ® 647 Mouse IgG1, κ BD Pharmingen (Cat# 557714, Lot# IsotypeIsotype Control 34876) control Mouse IgG2a κ Isotype ControleBiosciences (Cat# 11-4724-81, Isotype FTIC - Lot# E00590-1630) controlIsotype FITC Goat Anti mouse Ig (Cat# 611233, Lot# 039557) PE mouse IgGκ isotype control (Cat# 555749, Lot# 38193) Abbreviations: IHC,immunohistochemistry; ICC, immunocytochemistry; FC, flow cytometry; WB,Western Blot; ELISA, Enzyme-linked immunosorbent assay; PE,Phycoerythrin, APC, Allophycocyanin; FITC, Fluorescein isothiocyanate;HRP, horseradish peroxidase; CCSP, Clara cell secretory protein; PCNA,Proliferating cell nuclear antigen; SSEA4, Stage-Specific EmbryonicAntigen-4; Oct4, Octamer-binding transcription factor 4; Muc-1, Mucin 1;SPA, Surfactant protein A; SPB, Surfactant protein B; SPC, Surfactantprotein C; Nkx2.1, NK2 homeobox 1; Sox2, SRY (sex determining regionY)-box 2; Sox17, SRY-box 17; Tbx1, T-box 1; Pax 9, Paired box gene 9;CXCR4, C-X-C chemokine receptor type 4; FoxA2, forkhead box protein A2;AF647, Alexa Fluor ® 647.

Cultivation of Human iPS Cells.

The human iPS cell lines, iPSC (IMR90, C1) and iPSC (neonatal foreskin,C2), were obtained (Takahashi K, et al. 2007. Cell 131(5):861-872). Bothhuman iPS cell lines were generated by lentiviral transduction ofisolated human skin fibroblasts (IMR90, C1 clone) and neonatal foreskinfibroblast (C2 clone) with OCT-4, SOX2, Nanog and lin28 genes. Theseinduced pluripotent human stem cells have been extensivelycharacterized; they have normal karyotypes and telomerase activity,express cell surface markers and genes that characterize human ES cells,and maintain the developmental potential to differentiate into advancedderivatives of all three primary germ layers (Takahashi K, et al. 2007.Cell 131(5):861-872). Both lines were cultured and maintained asdescribed previously (Takahashi K, et al. 2007. Cell 131(5):861-872).Briefly, iPS cells were propagated on irradiated mouse embryonicfibroblast (MEF) feeder layers in DMEM-F12 media supplemented with 20%knock out serum replacement, 4 ng/ml bFGF, 1 mM glutamine, 1% mMnon-essential amino acids and 0.1 mM β-mercaptoethanol at 37° C., 5% CO2and 90-95% humidity, with medium changes every day. Undifferentiated iPScells were passaged every 4-5 days onto fresh feeders by mechanicaldissociation using a Stem Cell Cutting Tool (VWR).

In Vitro Differentiation of iPS Cells to AETII Cells

Human iPSCs were differentiated to alveolar epithelium in a directeddifferentiation protocol via definitive endoderm (DE) and anteriorforegut endoderm (AFE). iPS cells were differentiated towards definitiveendoderm under conditions described previously (Duan Y, et al. 2010.Stem Cells 28(4):674-686, Kubo A, et al. 2004. Development.131(7):1651-1662). Briefly, h-iPSC were cultured in RPMI 1640 mediumsupplemented with 100 ng/ml activin A, 2 mM L-glutamine and 1%antibiotic-antimycotic for 48 hours; 1×B27 supplement, 0.5 mM sodiumbutyrate and 0.1% FBS were added into the same medium and the cells werecultured for another 4 days, with daily medium changes (D'Amour K A, etal. 2005. Nat Biotechnol 23(12):1534-1541).

DE generated by exposure to activin A were trypsinized, reseeded at aratio of 1:1-2 on human ECM protein-coated plates and differentiated toanterior foregut endoderm with IMDM+5% FBS, 2 mM L-glutamine, 1 mMnonessential amino acids, 1% antibiotic-antimycotic supplemented with200 ng/ml NOGGIN and 10 mM SB-431542 for 2 days (Longmire T A, et al.2012. Cell Stem Cell 10(4):398-411, Green M D, et al. 2011. NatBiotechnol 29(3):267-272).

AFE cells were maintained in IMDM differentiation medium with 10% FBS, 2mM L-glutamine, 1 mM nonessential amino acids, 1%antibiotic-antimycotic, retinoic acid (0.5 μM), FGF-10 (10 ng/ml), EGF(10 ng/ml), Wnt3a (100 ng/ml), and KGF (10 ng/ml each) for 10-14 days.Cells were maintained in SAGM culture medium (Lonza), plus 1% fetalbovine serum (FBS) until seeding into lung matrices (Longmire T A, etal. 2012. Cell Stem Cell 10(4):398-411, Green M D, et al. 2011. NatBiotechnol 29(3):267-272).

Differentiated cells at day 22 were then maintained in DMEM medium with10% FBS, 2 mM L-glutamine, 1 mM nonessential amino acids, 1%antibiotic-antimycotic and 100 mM IWR-1 for 7 days.

Isolation of Human Type II Cells

Alveolar type II (AETII) cells were isolated from human lungs rejectedfor transplant as previously described (Bove P F et al. 2010. J BiolChem 285(45):34939-34949). Briefly, the right middle lobe was cannulatedthrough the main stem bronchus and removed from the rest of the lung.The distal airspaces were lavaged 6-10 times using a Ca²⁺- and Mg²⁺-freesolution (0.5 mm EGTA, 140 mm NaCl, 5 mm KCl, 2.5 mm Na₂HPO₄, 10 mmHEPES, and 6 mm glucose) and lavaged 3 times with a modified version ofthis solution (no glucose, 2.0 mm CaCl₂ and 1.3 mm MgSO₄). Elastase (13units/ml), was instilled into the distal airspaces and incubated at 37°C. for 30 min. Isolated cells were resuspended in DMEM and decanted ontoPBS- and DMEM-rinsed Petri dishes coated with human IgG antibody. After60 min at 37° C., non-adherent AETII cells were incubated with amonoclonal antibody against fibroblasts (AS02) and pan-mouse IgGDynabeads for removal by magnet. AETII cells were resuspended in DMEMcontaining 10% FBS, amphotericin B, ceftazidime, tobramycin, andvancomycin.

Flow Cytometry and Immunochemistry

DE, AFE and iPSC-AETII cell populations were assessed byimmunofluorescence or/and flow cytometry before differentiation, duringthe induction of DE and AFE and iPSC-AETII, and after cultivation in thedecellularized lung matrix.

For immunostaining, cells were washed with PBS, fixed in 4%paraformaldehyde for 20 min at room temperature (RT) and permeabilizedwith 0.1% Triton X-100 in PBS for 15 min at RT. Cells were blocked in 3%BSA in PBS for 60 min at RT and incubated with primary antibodyovernight at 4° C. The next day, cells were washed with PBS andincubated with secondary antibody for 2 h at RT. After washing, thecells were incubated with 4, 6-diamidino-2-phenylindole (DAPI) (1:1000)nuclear stain.

Paraffin sections of cell-seeded lung scaffolds were stained with H&E.Additional sections were permeabilized with 0.2% Triton X for 15 minafter heat-mediated citric acid antigen retrieval and blocked with 5%BSA for 1 hr. at RT. Primary antibodies were applied overnight at 4° C.Sections were incubated with secondary antibodies for 1 hr at RT,rinsed, treated with DAPI for 1 minute, and mounted with PVA-DABCO coverslipping solution. Stained cells and slides were imaged with a ZeissAxiovert 200M inverted microscope and a Hamamatsu camera.

For flow cytometry, cells were dissociated into single-cell suspensionsby incubation with 0.25% trypsin for 2 min, and fixed(Fixation/Permeabilization kit, BD Biosciences). After blocking for 30min on ice, the cells were incubated with primary antibody in blockingsolution for 30 min on ice. The cells were resuspended in 350 μl ofPerm/Wash buffer after incubation with conjugated secondaries for 30 minon ice, washed twice, and analyzed by flow cytometry. See Table 1 forantibody information.

Real Time Quantitative RT-PCR

Total RNA was extracted using the RNeasy Mini Kit from Qiagen, followingthe manufacturer's instructions. First-strand complementary DNA (cDNA)was synthesized with random hexamers as primers, using SuperScriptFirst-Strand Synthesis System according to manufacturer's protocol(Invitrogen). Each sample was run in triplicate with iQ™ SYBR GreenSupermix (Bio-Rad). PCR conditions included an initial denaturation stepof 4 min at 95° C., followed by 40 cycles of PCR consisting of 15 s at95° C., 30 s at 60° C., and 30 s at 72° C. Average threshold cycle (Ct)values from the triplicate PCR reactions for a gene of interest (GOI)were normalized against average GAPDH Ct values from the same cDNAsample. Fold change of GOI transcript levels between sample A and sampleB equals 2^(−ΔΔCt), where ΔCt=Ct_((GOI))−Ct_((GAPDH)), andΔΔCt=ΔCt_((A))−ΔCt_((B)). See Table 2 for primers.

TABLE 2 Sequences of primers used in qRT-PCR for various experimentsLength Gene (bp) Primer Sequences hSPA 180Forward: TCCAAGCCACACTCCACGA; (Seq id no: 1)Reverse: TTCCTCTGGATTCCTTGGG; (Seq id no: 2) hSPB  69Forward: TGGGAGCCGATGACCTATG; (Seq id no: 3)Reverse: GCCTCCTTGGCCATCTTGT; (Seq id no: 4) hNKX2.1  93Forward: GGACGTGAGCAAGAACATG; (Seq id no: 5)Reverse: TCGCTCCAGCTCGTACACC; (Seq id no: 6) hSPC  94Forward: CCTTCTTATCGTGGTGGTGGT; (Seq id no: 7)Reverse: TCTCCGTGTGTTTCTGGCTCAT; (Seq id no: 8) hMucin-1  88Forward: AGCTTCTACTCTGGTGCACAA; (Seq id no: 9)Reverse: GGTGGCTGGGAATTGAGA; (Seq id no: 10) hOCT4 164Forward: CCTCACTTCACTGCACTGTA; (Seq id no: 11) endogenousReverse: CAGGTTTTCTTTCCCTAGCT; (Seq id no: 12) hSOX2 151Forward: CCCAGCAGACTTCACATGT; (Seq id no: 13) endogenousReverse: CCTCCCATTTCCCTCGTTTT; (Seq id no: 14) hNANOG 239Forward: CCAAATTCTCCTGCCAGTGAC; (Seq id no: 15) endogenousReverse: CACGTGGTTTCCAAACAAGAAA; (Seq id no: 16) hCC10 105Forward: CCCTGGTCACACTGGCTCTC; (Seq id no: 17)Reverse: TCATAACTGGAGGGTGTGTC; (Seq id no: 18) hCXCR4  79Forward: CACCGCATCTGGAGAACCA; (Seq id no: 19)Reverse: GCCCATTTCCTCGGTGTAGTT; (Seq id no: 20) hFOXA2  89Forward: GGGAGCGGTGAAGATGGA; (Seq id no: 21)Reverse: TCATGTTGCTCACGGAGGAGTA; (Seq id no: 22) hSOX17  61Forward: GGCGCAGCAGAATCCAGA; (Seq id no: 23)Reverse: CCACGACTTGCCCAGCAT; (Seq id no: 24) hPAX9 132Forward: GTTATGTTGCTGGACATGGGT; (Seq id no:25)Reverse: GAAGCCGTGACAGAATGACTAC; (Seq id no: 26) hTBX1 117Forward: GCTCCTACGACTATTGCCC; (Seq id no: 27)Reverse: CGTATTCCTTGCTTGCCCT; (Seq id no: 28) hCD31 140Forward: ATTGCAGTGGTTATCATCGGAGTG; (Seq id no: 29)Reverse: CTCGTTGTTGGAGTTCAGAAGTGG; (Seq id no: 30) hTSHR 156Forward: TTTCTTACCCAAGCCACTGC; (Seq id no: 31)Reverse: TTCTCTTCATATTCCTGGTGG; (Seq id no: 32) hALB 149Forward: AAACGCCAGTAAGTGACAGAG; (Seq id no: 33)Reverse: ATATCTGCATGGAAGGTGAAT; (Seq id no: 34) hGAPDH 122Forward: GACAACAGCCTCAAGATCATCAG; (Seq id no: 35)Reverse: ATGGCATGGACTGTGGTCATGAG; (Seq id no: 36)

Transmission Electron Micrograph

Cell samples were prepared following a modified protocol from Schmiedlet al (Schmiedl, et al. Histochem Cell Biol 1-12). Briefly, native humanAETII and iPSC-AETII cells were fixed at 37° C. with a 2.5%glutaraldehyde/2.0% paraformaldehyde mixture in 0.2M sodium cacodylatefor 30 minutes, followed by 2 hour incubation at 4° C. The samples weredehydrated following a standard ethanol series. The samples werepost-processed by OsO₄ fixation and en block uranyl acetate staining.Sections (70 to 80-nm) were taken and incubated in uranyle acetate andlead citrate for increased contrast. Images were taken using a PhilipsTecnai transmission electron microscope.

Preparation of Decellularized Extracellular Matrix Scaffolds

Three-month-old Fischer or Sprague Dawley rats were anesthetized withsodium pentobarbital, according to the guidelines set forth by theAmerican Veterinary Medical Association (60 mg/kg IP). Lungextracellular matrix scaffolds were prepared as previously described(Petersen T H, et al. 2010. Science 329(5991):538-541, Calle E A, et al.2011. J Vis Exp (49). pii: 2651). Lungs were perfused with heparin (50U/ml, Sigma) in PBS, and removed with the heart and trachea. Thepulmonary artery and trachea were cannulated and the lungs were perfusedthrough the pulmonary artery with sodium nitroprusside (1 ml/ml, Fluka)before being treated with decellularization solution (8 mM CHAPS, 1MNaCl, 5 mM EDTA in PBS) for 2-3 hours at 37° C. Scaffolds were treatedwith benzonase endonuclease (90 U/ml, Sigma) for 1 hr. at 37° C.,followed by extensive rinsing with PBS, antibiotics and antimycotics.

Human lungs were obtained from beating-heart donors or warm autopsy asarranged through Gift of Life Michigan, and were decellularized asrecently described (Booth A J. 2012. Am J Resp Crit Care Med. 186(9):866-76). Lung samples were agitated in sterile deionized, distilledwater and incubated in 0.1% Triton X-100 for cell lysis. Samples werewashed with sterile PBS, incubated with 2% sodium deoxycholate andwashed again. Lungs were incubated in 1M NaCl to lyse residual nuclei.After decanting NaCl, tissues were rinsed and incubated with 30 μg/mLDNAse in 1.3 mM MgSO4 and 2 mM CaCl2. The DNAse solution was decantedand tissues were washed with sterile PBS. (Booth A J, et al. 2012. Am JResp Crit Care Med. 186(9): 866-76).

Culture of Cells on Rat Lung Extracellular Matrix Scaffolds

Rat scaffolds were mounted in the bioreactor as described previously(Petersen T H, et al. 2010. Science 329(5991):538-541). Cannulas wereconnected to tubing loops to provide perfusion and introduction of cellsto the scaffold. Forty million iPSC-AETII cells were suspended in 3-5 mlof culture medium (SAGM-1% FBS) and introduced into the airwaycompartment; perfusion was initiated at 1 ml/min immediately after cellseeding. In additional experiments, 6×10⁶ native human AETIIcells—isolated from human lung—were introduced into the upper right lobeof a decellularized rat lung. The full volume of culture media waschanged once at day 3 or 4 and samples were harvested at days 1, 3 and 7and saved for histology. In parallel experiments, iPSC-AETII cells wereseeded onto sections of decellularized rat lung at a concentration of1.5×10⁵ cells/slice in SAGM-1% FBS media for 7 days. Finally, 3×10⁵ ofeither AETII cells or native human AETII cells were transferred ontodecellularized human lungs slices in SAGM-1% FBS and cultured for 1week; media was changed every other day.

Enzyme-Linked Immunosorbent Assay Analysis (ELISA) for SPC

ELISA was performed on cell culture media collected during iPSC-AETIIdifferentiation to quantify secreted SPC (Life Science AdvancedTechnology) according to the manufacturer's instructions. SPC valueswere normalized to the total number of cells.

Western Blotting

Cells were incubated in RIPA buffer supplemented with proteaseinhibitors (Complete Mini, Roche,) on ice for 30 min. Proteinconcentration was determined from cell lysates using a bicinchoninicacid protein assay (Thermo Fisher Scientific). Cell lysates weredenatured and equal amounts of protein per sample were subjected toSDS-PAGE and immunoblotting as described previously. HRP-conjugated goatanti-mouse and goat anti-rabbit secondaries were detected by enhancedchemiluminescence.

Proliferation Assay

To assess cell proliferation within the lung scaffold, lungs seeded andcultured with AETII for 3 days and 7 days were fixed in 4% PBS-bufferedparaformaldehyde (pH 7.4) and post-fixed with 70% EtOH. Theimmunocytochemical staining against human caspase and PCNA was performedas described in elsewhere herein. The images were visualized with aZeiss Axiovert 200M inverted microscope and imaged with Hamamatsucamera. The percentage of positive nuclear staining was calculated basedon total cell numbers in three high power fields.

Statistical Analyses

Statistics were done with Origin (OriginLab, Northampton, Mass.). Thedata were expressed as mean±SEM. (standard error of measurement, allerror bars represent ±SEM). Unpaired, two-tailed Student's t-tests wereperformed to evaluate whether the two groups were significantlydifferent from each other. p values less than 0.05 (two-tailed) wereconsidered statistically significant. All error bars represent ±SEM

The results of the experiments are now described.

The experiments presented herein demonstrate an efficient andconsistent, step-wise differentiation method to generate definitiveendoderm (DE), anterior foregut endoderm (AFE), and subsequently, arelatively homogeneous population of human AETII and AETI cells fromhuman iPSCs (iPSCs) (FIG. 1A). These cells not only demonstrate thephenotype of mature human alveolar type I and type II cells, but alsoexpress a high percentage of type I and II cell markers when compared tofreshly isolated human primary alveolar type I and type II cells.Additionally, these iPSC-derived AETII cells are capable of repopulatingan acellular lung matrix, and give rise to cell types that reside in thedistal lung (FIG. 1B).

Efficient Derivation of Definitive Endoderm Cells

Embryonic lung arises from definitive endoderm (DE) (Green M D, et al.2011. Nat Biotechnol 29(3):267-272, Banerjee E R, et al. 2012. PLoS One7(3):e33165, Kadzik R S, et al. 2012. Cell Stem Cell 10(4):355-361).Therefore, in the first step, iPSC were differentiated to DE by exposingthem to saturating concentrations of activin A during the first 6 daysof differentiation. iPSC were initially cultured without serum for 48hours with 100 ng/ml activin A, and then changed to a low serumconcentration provided with 1×B27 culture medium. During the time thatiPSC were exposed to activin A, the majority of the cells in thecolonies converted to DE cells, while those cells that did not graduallydied as monitored through visual observation. After 6 days ofdifferentiation, both iPSC clones (denoted as C1 and C2) stainedpositively for SOX17 and FOXA2, and the majority of the cells werepositive for both SOX17 and FOXA2 (FIG. 1D, FIG. 7A-7J for C1 cells,FIG. 8A-8J for C2 cells). When endoderm marker expression was monitoredusing qRT-PCR for SOX17, CXCR4 and FOXA2, no expression of these markerswas observed at day 0; expression then increased from day 0 to day 6 iniPS cells exposed to activin A (FIG. 7L and FIG. 12A for C1 cells, FIG.8L and FIG. 12B for C2). Flow cytometric analysis demonstrated that thecell population derived from iPS cells at day 6 expressed a highpercentage of markers associated with definitive endoderm, including92.71±4.0% for CXCR4, 83.7 6±2.0% for SOX17, and 87.66±1.2% FOXA2 in C1and 87.23±2.0% for CXCR4, 91.42±3.0% for SOX17, and 83.54±1.8% FOXA2 inC2 (FIG. 7M for C1 cells, FIG. 8M for C2). It was found that theprotocol used herein was highly efficient for generating a relativelyhomogeneous population of DE from iPSC; based on the dual expression ofSOX17 and FOXA2 in iPSC clones. It was observed that more than 85% of C1and 89% of C2 was comprised of endodermal cells (FIG. 7K for C1 cells,FIG. 8K for C2). Both the C1 clone (which is of fetal lung origin) andthe C2 clone (which is derived from neonatal fibroblasts) yield similarresults. This suggests that this protocol may be generalized to otheriPSC lines from other cell origins or that are reprogrammed using othertechniques.

Generating Anterior Foregut Endoderm from Definitive Endoderm Cells

Following developmental paradigms, directed differentiation of iPS cellsto alveolar epithelium should proceed by generation of definitiveendoderm, followed by patterning into anterior foregut endoderm (AFE).Differentiation of AFE cells from DE was induced by exposing cells toNOGGIN (200 ng/ml) and SB-431524 (10 mM) for 2 days, per the conditionsdescribed previously by Green and colleagues (Longmire T A, et al. 2012.Cell Stem Cell 10(4):398-411, Green M D, et al. 2011. Nat Biotechnol29(3):267-272, Mou H, et al. 2012. Cell Stem Cell 10(4):385-397).Application of NOGGIN/SB-431542 to definitive endoderm yielded a highlyenriched population of cells with strong expression of markersassociated with the AFE phenotype, including SOX2, PAX9 and TBX1 in bothclones. The majority of cells co-expressed SOX2 and FOXA2, asdemonstrated by immunostaining at day 8 (FIG. 2A-FIG. 2J for C1 cells,FIG. 9A-FIG. 9J for C2). Cells that were negative for definitiveendoderm markers gradually died off after switching to AFEdifferentiation media as we visualized by microscopy. In addition, thepresent results show that inhibition of TGF-β signaling and activinA/nodal signaling with NOGGIN/SB-431542 was sufficient in the two iPSCclones tested to increase the anterior endoderm cell population (asdefined by FOXA2⁺/SOX2⁺) up to 92-95% as compared to <0.1% withoutNOGGIN/SB-431542. (FIG. 2K, for C1 cells, FIG. 9K for C2). QuantitativeRT-PCR revealed a relatively modest increase in both PAX9 and TBX1 whencompared to SOX2 expression, which was highly expressed in AFE cellsderived from both iPSCs colonies at day 8 (FIG. 2L, for C1 cells, FIG.9L for C2). After activin A removal at day 6 and switching toNOGGIN/SB-431542, it was observed that both CXCR4 and SOX17 decreasedfrom day 6 to day 12. In the case of FOXA2, an increase in expression atday 8 was observed, followed by a decrease with time in culture (FIG.12A for C1 cells, FIG. 12B for C2). These results are expected, giventhat endoderm is a transient stage in lung development and is expectedto peak and then fade as stem cells differentiate toward laterphenotypes (Yasunaga M, et al. 2005. Nat Biotechnol 23(12):1542-1550).

Prior to lung differentiation, all cells that will belong to thepulmonary lineage must first progress through a primordial progenitorstage defined by the upregulation of the ventral marker, NKX2.1(Longmire T A, et al. 2012. Cell Stem Cell 10(4):398-411, Van Haute L,et al. 2009. Respir Res 10:105). NKX2.1 (homeodomain-containingtranscription factor) is the earliest known marker associated withcommitment to thyroid and lung, but several studies suggest that NKX2.1induction is indicative of commitment to a lung, rather than a thyroidfate (Longmire T A, et al. 2012. Cell Stem Cell 10(4):398-411, Green MD, et al. 2011. Nat Biotechnol 29(3):267-272, Van Haute L, et al. 2009.Respir Res 10:105). Replacing activin A with NOGGIN/SB-431542 from day 6to day 8, followed by the addition of a cocktail containingBMP4/Wnt3a/bFGF/KGF induced NKX2.1 expression in the AFE cell populationat day 13. Most of NKX2.1 positive cells in both iPSC clones co-stainedwith the endoderm marker FOXA2 (FIG. 2M and FIG. 2N for C1 cells, FIG.9M and FIG. 9N, for C2). Additionally, flow cytometric analysis showedthat 24±2% of the AFE cell population derived from C1 and 26±3% from C2was positive for NKX2.1, as compared to <1% in activin A induced cellsand in cells cultured in media without NOGGIN/SB-431542 at day 13.Continuous activin A treatment from day 6 to day 8 without the additionof NOGGIN/SB-431542 resulted in rare FOXA2⁺/SOX2⁺ cells and few NKX2.1⁺cells (FIG. 2O, for C1 cells, FIG. 9O for C2). Collectively, theseexpression data show that in activin A-induced definitive endoderm,exposure to NOGGIN/SB-431542 results in a highly enriched population ofcells with an AFE phenotype.

Extracellular Matrix Protein Effects on Differentiation

In traditional stem cell cultivation/differentiation experiments, growthfactors (GFs) are added in soluble form in order to provide signals fortissue-specific differentiation (Green M D, et al. 2011. Nat Biotechnol29(3):267-272, Mou H, et al. 2012. Cell Stem Cell 10(4):385-397, Ali NN, et al. 2002. Tissue Eng 8(4):541-550, Rippon H J, et al. 2006. StemCells 24(5):1389-1398, Banerjee E R, et al. 2012. PLoS One 7(3):e33165).However, differentiation has recently become increasingly linked tomechanobiological concepts such as interaction between cells and theextracellular matrix (ECM) (Reilly G C, et al. 2010. J Biomech43(1):55-62, Lin Y M, et al. 2010. Tissue Eng Part A 16(5):1515-1526,Gutierrez J A, et al. 1998. Am J Physiol 274(2 Pt 1):L196-202).Moreover, during development, ECM protein expression represents some ofthe most important inducers of organ fate. Accordingly, before switchingmedia to NOGGIN/SB-431542 to generate AFE from human pluripotent cells,the DE cells were split with trypsin and reseeded at a ratio of 1:1-2 onECM-coated plates in media containing NOGGIN/SB-431542 for 48 hours.Adhesion of DE cells to fibronectin, collagen I, collagen IV, Matrigel,and a mixture of human ECM proteins (comprising of collagens, laminin,fibronectin, tenascin, elastin, and a number of proteoglycans andglycosaminoglycans; Sigma) was examined. Fibronectin, collagen I,collagen IV and laminin are principal components of lung matrix, and DEcells attach well to all of these proteins. However, mixed human ECMprotein resulted in faster DE cell attachment and significantly higherexpression of SPC, SPB, and NKX2.1 genes on both day 15 and 30 (FIG.10A-FIG. 10C).

Efficient Derivation of Purified Lung Alveolar Type II from AFE

After differentiation to AFE on day 8, the medium was switched toalveolar differentiation medium containing FGF-10, EGF, WNT3a, KGF, andRA for 14 days on human ECM protein. These factors or reagents werechosen through empiric studies, and are thought to play a crucial rolein alveolar pneumocyte differentiation and lung development (Longmire TA, et al. 2012. Cell Stem Cell 10(4):398-411, Green M D, et al. 2011.Nat Biotechnol 29(3):267-272, Mou H, et al. 2012. Cell Stem Cell10(4):385-397, Ali N N, et al. 2002. Tissue Eng 8(4):541-550, Rippon HJ, et al. 2006. Stem Cells 24(5):1389-1398, Banerjee E R, et al. 2012.PLoS One 7(3):e33165). Compared with other reports, it was found thatthe differentiation cocktail that lacked BMP4 in the final stage,resulted in distal markers, especially those associated with type IIpneumocytes. After day 22, the cells—now termed AETII cells (FIG. 1A andFIG. 1C)—were maintained in SAGM culture medium containing 1% FBS. AETIIcells derived from both the C1 and C2 clones were strongly positive fortype II markers, including pro-SPC, pro-surfactant protein B (SPB),mucin-1 and surfactant protein A (SPA). In addition to the positivemarker expression associated with type II cells, the presence oflamellar bodies, typical of human type II cells, was also examined byelectron microscopy in the iPSC-derived AETII cells. TEM identificationof lamellar bodies is used as a method for positively identifying type 2pneumocytes. The TEM data clearly show the presence of lamellar bodiesin the iPSC-derived AETII cells. (FIG. 3A-FIG. 3F, for C1 cells, FIG.11A-FIG. 11F). Quantitative RT-PCR demonstrated a high percentage ofexpression of type II cell markers in iPSC-AETII cells, that wascomparable to expression levels of freshly isolated human primaryalveolar type II cells (hATII cells) (FIG. 3G, for C1 cells, FIG. 11Gfor C2). Up to 97% of cells were positive for SPC, 92±0.9% positive forMucin-1, 89±0.9% positive for SPB and the vast majority of the cells,94±0.9%, expressed the epithelial surface marker CD54 in clone C1 and96.27±0.5% for SPC, 94.42±0.3% for SPB, and 91.54±1.8% for Mucin-1 and89.83±0.5% for CD54 in clone C2. Moreover, AETII cells were negative forCCSP (a Clara cell marker), p63 (basal stem cell marker) and SOX2(proximal airway epithelial cell marker) by FACS analysis, indicatingthat these cells are a relatively homogeneous population of type IIcells (FIG. 3H and FIG. 3I, for C1 cells, FIG. 11H and FIG. 11I for C2).ELISA measurements of SPC protein in the cell culture supernatantsindicated that day 13 cells synthesized and secreted SPC at a rate of16.78 ng/ml and 15.78 ng/ml in C1 and C2 clones, respectively, every 24h. The rate of secretion was significantly increased on day 22 ofdifferentiation when compared to secretion at day 13 (P<0.05). At day22, the iPSC-AETII cells from C1 and C2 produced 68.5 ng/ml and 71.5ng/ml SPC every 24 h, which was comparable to that produced by freshlyisolated human AETII cells (82.95 ng/ml) (FIG. 3J, for C1 cells, FIG.11J for C2).

A previous study has found that airway progenitor cells, whichultimately give rise to trachea, bronchus and bronchioles, areNKX2.1⁺/SOX2⁺ and sustain high levels of SOX2 expression (Mou H, et al.2012. Cell Stem Cell 10(4):385-397). However, by day 22, noNKX2.1⁺/SOX2⁺ cells or single positive SOX2⁺ cells were detected in thepopulation of differentiated iPSC-AETII cells. All the AETII cells werealso negative for CCSP and p63 as determined by flow cytometry (FIG. 3Hfor C1 cells, FIG. 11H for C2 cells). This may indicate that these cellsare not airway progenitor cells. Since anterior foregut endoderm can betheoretically differentiated into cells expressing markers of thyroid,parathyroid and lung (Longmire T A, et al. 2012. Cell Stem Cell10(4):398-411), the expression of CD31 (endothelial marker), albumin(mature hepatocyte marker) and TSGHR (thyroid cells marker) wereinvestigated by quantitative RT-PCR, to determine whether the iPSC-AETIIcells were contaminated by cells from these other lineages. No specificmarkers of thyroid, endothelial cells or hepatocyte lineage weredetected in iPSC-AETII cells at day 22, thereby confirming the absenceof cells from these lineages in the differentiated lung progenitorpopulation (FIG. 3I, for C1 cells, FIG. 11I for C2 cells).

The expression of AFE markers (SOX2, PAX9 and TBX1) and DE markers(SOX17, CXCR4 and FOXA2) decreased from day 8 to day 22. By day 32, noneof these markers were detectable in iPSC-AETII cells. In case of FOXA2and SOX2, after activin A removal at day 6 and switching toNOGGIN/SB-431542 an increase in FOXA2 and SOX2 expression was observedat day 8 followed by a decrease in the expression of these genes inculture over time (FIG. 12A, FIG. 12C, and FIG. 16B for C1 cells, FIG.12B, FIG. 12D, and FIG. 16CC for C2 cells).

Following exposure to alveolar pneumocyte induction media containingWnt3a, EGF, KGF and FGF at day 9 of culture, there was lower expressionof AFE genes, especially SOX2, and there was a concomitant upregulationof NKX2.1. After day 8, SOX2 was rapidly downregulated while NKX2.1 wasupregulated. The expression of NKX2.1 gradually increased from 24±2% atday 13 to 94±0.4% at days 20-22 in C1. Quantitative RT-PCR and flowcytometry revealed that the percentage of SPC positive cells graduallyincreased from 58±1.6% at day 13 to 90±0.4% at days 20-22 duringdifferentiation in the C1 clone. The same pattern of NKX2.1 and SPCexpression was observed in the C2 clone during differentiation from day8 to day 22. (FIG. 13A-FIG. 13D, for C1 cells, FIG. 14A-FIG. 14D for C2cells). Between days 8 and 22, NKX2.1⁺ cells proliferated slowly,ultimately leading to an increase in the number of NKX2.1 and SPCpositive cells. The amount of cell death, following the switch inculture medium to alveolar epithelium differentiation medium, wasnegligible when compared to the earlier medium switch from DE to AFEdifferentiation medium from day 0 to day 8. Immunostaining for NKX2.1⁺cells at different days (day 13, 18 and 21) demonstrated that coloniesexpressing NKX2.1 were gradually expanded and over-grew the NKX2.1negative cells. This ultimately led to an increase in the number ofNKX2.1 positive cells, as shown by a gradual progression from 24% at day13 to 94% at days 20-22.

Since several recent studies have reported CD166 and α6β4 to be markersof lung epithelial progenitors (Chapman H A, et al. 2011. J Clin Invest121(7):2855-2862, Whitsett J A, et al. 2011. J Clin Invest121(7):2543-2545, Soh B S, et al. 2012. Mol Ther 20(12):2335-2346,Asselin-Labat M L, et al. 2012. Open Biol 2:120094), the expression ofthese markers were characterized during the differentiation of iPSC toepithelial cells at several time points. Flow cytometry revealed thatthe percentage of CD166 positive cells gradually increased from 15±0.8%at day 13 to 35±1.4% at days 20-22 during differentiation in the C1clone. The expression of CD166 subsequently decreased in mature culturesof iPSC-derived epithelial cells at day 28. In the case of α6β4, theexpression progressively increased from 10±0.7% at day 13 to 25±1.2% atday 18 in C1. By day 22 only 7-8% of the cells were positive for α6β4.The same pattern of CD166 and α6β4 expression was observed in the C2clone during differentiation from day 8 to day 22. (FIG. 15A and FIG.15B, for C1 cells, FIG. 15C and FIG. 15D for C2 cells).

Replacing the NOGGIN/SB-431542 media with differentiation media at day 9of culture resulted in decreasing expression of pluripotency genes suchas OCT4 and Nanog over time. As expected, from day 13 to day 22 thepluripotency gene expression was almost undetectable. Flow cytometry oniPSC-derived AETII cells from both clones showed that SPC-positive cellsat day 22 were negative for OCT4 (FIG. 16A-FIG. 16E for C1 and C2cells).

Since type II cells can spontaneously differentiate into cellsexpressing markers of type I cells (Fehrenbach H, et al. 2001. RespirRes 2(1):33-46, Bove P F et al. 2010. J Biol Chem 285(45):34939-34949,Fujino N, et al. 2011. Lab Invest 91(3):363-37833) the expression oftype I markers, T1α, AQ5, and caveolin-1, were assessed by flowcytometry and qPCR. Flow cytometry revealed that 8-11% of cellsexpressed type I markers following the differentiation protocol. Up to9% of cells were positive for AQ5, 9.6% positive for caveolin-1 and 8.1%expressed the type I surface marker T1a in clone C1 (FIG. 4C).

Differentiation of iPSC-Derived AETII to Type I Cells

To further differentiate iPSC-AETII to AETI, we next examined the effectof modulating Wnt/β-catenin signaling on type I marker expression inAETII cells. We examined whether the selective β-catenin/CBP inhibitorIWR-1 (Banerjee E R, et al. 2012. PLoS One 7(3):e33165, Fehrenbach H, etal. 2001. Respir Res 2(1):33-46) would modulate the differentiation ofiPSC-AETII cells to AETI cells. Differentiated AETII cells at day 22were cultured on human ECM protein coated plates in DMEM-10%, FBSsupplemented with 100 mM IWR-1 for 7 days. As data from independentexperiments indicated, incubation of iPSC-AETII cells with IWR-1 inducedthe differentiation of AETII cells to the AETI cell phenotype (FIG.4A-FIG. 4D). Following treatment with IWR-1, there was a significantincrease in AETI markers AQ5, T1α and caveolin-1, in iPSCs-derived AETIIcompared to untreated AETII as determined by immunostaining (FIGS. 4Aand 4B). Flow cytometry revealed that up to 92% of cells were positivefor aquaporin-5 (AQ5), 98% positive for caveolin-1, and 88% of the cellsexpressed the epithelial marker T1α (FIG. 4C). In contrast, the AETIIcell marker, SPC, decreased significantly as determined by flowcytometry (FIG. 4C). Quantitative RT-PCR demonstrated a high percentageof expression of type I cell markers in iPSC-AETII cells exposed toIWR-1, that was comparable to expression levels of freshly isolatedhuman primary alveolar type I cells (hAETI cells) (FIG. 4D)

Repopulation of Rat and Human Acellular Matrix with iPSC-Derived AETII

To explore the regenerative potential of iPS-derived AETII cells togenerate lung tissue in vitro, lungs from adult humans and rats weredecellularized by processes that remove cellular components but leavebehind a scaffold of extracellular matrix that retains the hierarchicalbranching structures of airways and vasculature (Petersen T H, et al.2010. Science 329(5991):538-541, Booth A J, et al. 2012. Am J Resp CritCare Med. 186(9): 866-76). This is an assay that was recently developedto test the regenerative potential of primary lung epithelial cells orstem cells derived lung epithelial cells (Longmire T A, et al. 2012.Cell Stem Cell 10(4):398-411, Petersen T H, et al. 2010. Science329(5991):538-541, Daly A B, et al. 2012. Tissue Eng Part A18(1-2):1-16, Ott H C, et al. 2010. Nat Med 16(8):927-933). Both ATI andATII pneumocytes are differentiated cell types, however several reportshave demonstrated that ATII cells retain a level of plasticity. Whendamage occurs to the ATI pneumocytes, ATII cells proliferate and can, inturn, differentiate into ATI cells (Asselin-Labat M L, et al. 2012. OpenBiol 2:120094, Fehrenbach H, et al. 2001. Respir Res 2(1):33-46, FujinoN, et al. 2011. Lab Invest 91(3):363-37833). Therefore, in the studiespresented herein the capacity of iPSC derived type II cells torepopulate the airway compartment of a decellularized lung extracellularmatrix was examined. iPSC-AETII cells at day 28 were utilized torepopulate the acellular matrices. This stage seemed most suitablebecause of their commitment to a pneumocyte type II phenotype andfunction. The seeded matrix was cultured in a bioreactor that wasdesigned and described previously (Petersen T H, et al. 2010. Science329(5991):538-541, Petersen T H, et al. 2011. Cell Transplant20(7):1117-1126). This bioreactor is capable of replicating key aspectsof the in vivo fetal lung environment, including vascular perfusion andliquid ventilation. In addition to seeding iPSC-AETII cells intodecellularized whole rat lung tissue, we also seeded iPSC-AETII cellsonto slices of either rat or human acellular lung matrix cultured in6-well plates (FIG. 1C)

For the rat lung bioreactor experiments, approximately 40×10⁶ cells wereinjected into the airway through the trachea to repopulate thedecellularized rat lung matrix (FIG. 1C). The seeded matrix was thencultured and maintained for up to 7 days in a bioreactor in SAGM (1%FBS). H&E staining showed that iPSC-AETII cells were able to diffuselyrepopulate alveolar lung structures within distal lung. The majority ofseeded AETII cells still showed approximate AETII morphology—a cuboidalshape and round single nuclei (FIG. 5A-FIG. 5C for C1 cells, FIG.17A-FIG. 17C for C2 cells). Many AETII cells within the lung matrixexpressed the type II cell marker, pro-SPC and NKX2.1. In native lung,this marker is normally found on AETII cells. In the reseeded rat lungs,cellular expression of pro-SPC was robust in the alveoli by day 3 andremained present at day 7 (FIG. 5D-5I for C1 cells, FIG. 17D-17I for C2cells).

It was investigated whether iPSC-derived AETII are able to proliferatein the acellular matrix. After culturing iPSC-AETII cells in thebioreactor, sections from the reseeded lung at day 3 and day 7 werestained for PCNA (proliferating cell nuclear antigen) expression. Themajority of cells on the rat scaffold expressed PCNA, at both day 3 andday 7 while they displayed few markers of apoptotic cell death, asdetermined by immunostaining for caspase-3. However, iPSC-AETII culturedin the rat lung scaffold for 7 days had an increased rate of positivePCNA staining when compared to the day 3 cultures. The data suggest thatthe iPSC-AETII are able to proliferate when they are seeded in ratscaffold (FIG. 5J-5N for C1 cells, FIG. 17-17M for C2 cells).

The formation of type 1 cells in vivo, from type II cells, isaccompanied by a loss of the NKX 2.1 protein (Longmire T A, et al. 2012.Cell Stem Cell 10(4):398-411). Consistent with this pattern, someengrafted AETII cells acquired a flattened morphology, and expressed thetype 1 pneumocyte marker T1α but lacked expression of NKX2.1 protein byco-staining (FIG. 5O). Moreover the number of T1α positive cellssignificantly increased from 9.7% before cell seeding to 31.2% afterculturing cells in the rat lung bioreactor. Conversely, the number ofSPC positive cells decreased from 98% before cell seeding to 72.3% after7 days cultured in rat lung scaffold in bioreactor. Since type I cellsare terminally differentiated cells and are not able to proliferate liketype II cells, These observations may indicate that the extracellularmatrix cues support a epithelial populations, and that iPSC-AETII areable to differentiate to type I cells within the lung scaffold. (FIG.5P). All of the iPSC-AETII cells were negative for CCSP and p63 by FACSanalysis after 7 days cultured in the rat lung scaffold (FIG. 5P).

In parallel experiments, iPSC-derived AETII were cultured on sections ofrat and human lung matrix. To prepare sections of the acellular lungmatrix, lungs from adult donors were treated using a procedure similarto that previously described (Petersen T H, et al. 2010. Science329(5991):538-541, Booth A J, et al. 2012. Am J Resp Crit Care Med.186(9): 866-76). Then, iPSC-AETII cells were seeded in the acellularmatrices by directly pipetting onto sections of the matrix (thickness:600 μm), and maintaining in culture in 6-well plates in SAGM with 1%FBS. The iPSC-AETII cells adhered well to matrix surfaces and wereinitially distributed widely throughout alveoli in the matrix, in boththe rat and human decellularized lung sections. Many of the cells stillshowed AETII morphology and there was robust expression of SPC andNKX2.1 (FIG. 6A-FIG. 6F on human lung sections and FIG. 6H-6K on ratlung sections), while some engrafted AETII cells acquired a flattenedmorphology and expressed the alveolar type I marker, T1a (FIG. 6C onhuman lung sections and FIG. 6J on rat lung section). iPSC-derived AETIIare able to proliferate on both rat and human lung sections, asdetermined by immunostaining for PCNA and caspase-3 (FIG. 6G on humanlung sections and FIG. 6L-FIG. 6O on rat lung section).

As a control for these experiments, isolated human type II cells fromadult human lung (hAETII cells) were also seeded onto decellularizedhuman lung sections. As with the iPSC-AETII cells, many of the cellswith hAETII morphology expressed SPC and NKX2.1, and some of the hAETIIcells gave rise to T1a positive cells (FIG. 6P-FIG. 6U). Moreover theywere able to replicate on the human scaffold sections, and the majorityof cells expressed PCNA, while they displayed few markers of apoptoticcell death, as determined by immunostaining for caspase-3 (FIG. 6V).

Differentiation of iPSCs Toward DE, AFE, AETII and AETI Cells

Lung epithelia remain among the least-studied lineages to be derivedfrom ESCs and iPSCs in vitro to date, and few research groups havereported on the differentiation toward lung epithelium (Wang D, et al.2007. Proc Natl Acad Sci USA. 104(11):4449-4454, Green M D, et al. 2011.Nat Biotechnol 29(3):267-272, Mou H, et al. 2012. Cell Stem Cell10(4):385-397, Van Haute L, et al. 2009. Respir Res 10:105). Conditionsfor directing hESCs or iPSCs to differentiate along an alveolarepithelial lineage with homogeneity are not yet fully defined, and mostprotocols generate a mixed population of alveolar epithelium from hESCsor iPSCs. In the mouse, a NKX2.1:GFP reporter was used to isolate cellscommitted to the lung fate which were then amenable to furtherdifferentiation (Longmire T A, et al. 2012. Cell Stem Cell10(4):398-411). Moreover, in heterogeneous cultures of differentiatingESCs, induction of late markers of development such as surfactantprotein C (SPC) have been reported, but their expression appears to bestochastic, and the cells expressing these markers have been difficultto expand (Longmire T A, et al. 2012. Cell Stem Cell 10(4):398-411,Green M D, et al. 2011. Nat Biotechnol 29(3):267-272, Mou H, et al.2012. Cell Stem Cell 10(4):385-397, Ali N N, et al. 2002. Tissue Eng8(4):541-550, Rippon H J, et al. 2006. Stem Cells 24(5):1389-1398,Banerjee E R, et al. 2012. PLoS One 7(3):e33165). The studies presentedherein demonstrate an efficient and consistent, step-wisedifferentiation method to generate definitive endoderm (DE), anteriorforegut endoderm (AFE), and subsequently, a relatively homogeneouspopulation of human alveolar type II cells from two different humaniPSCs clones (C1, reprogrammed from fetal lung fibroblasts and C2reprogrammed from neonatal fibroblasts). Interestingly both iPSC clonesyield similar results and had similar efficiency to differentiate towardDE, AFE, AETII and AETI cells, suggesting this protocol can begeneralized to other iPSC lines from other sources. Unlike isolatedhuman type II cells, however, these iPSC-AETII cells are capable ofproliferating for several passages without losing AETII cell-associatedmarkers, such as SPC, SPA and mucin-1, and can be used to generate tensof millions of cells with which to seed the acellular matrix scaffold.The ability to “scale up” a progenitor population will be particularlyvaluable when translating these technologies for use in producing humantissues, and allows for the possibility of using autologous iPSC derivedcells in future lung bioengineering work.

Under appropriate culture conditions, iPSC-derived AETII cells seededinto either rat decellularized lung bioreactors, or onto decellularizedrat or human lung sections, behave similarly to isolated human type IIcells. In these studies, the iPSC-AETII cells were able to diffuselyrepopulate alveolar lung structures. Although the majority of seedediPSC-AETII cells still showed approximate AETII morphology, thepercentages of T1α positive cells increased to approximately 30% withinthe rat lung matrix over a 7-day culture period. While not wishing to bebound by any particular theory, it is possible that this shift inexpression is a differentiating effect of correct cell-matrixinteractions. Although type II cells are differentiated cells, thesecells nonetheless retain a level of plasticity. Following peripherallung injury, type II cells undergo proliferation and differentiationtoward the type I phenotype. In fact, type II cells are considered to beputative alveolar stem cells and are crucial to the natural regenerativeprocess of the alveoli (Banerjee E R, et al. 2012. PLoS One 7(3):e33165,Asselin-Labat M L, et al. 2012. Open Biol 2:120094). The interactionbetween AETII cells and the lung matrix may have been especiallyimpactful since it is likely that the AETII cells are still in aprogenitor state able to give rise to T1α cells when they adhere inregions where native lung contains type I cells and maintain a type IIcell phenotype in regions where type II cells are typically be found.While not wishing to be bound by any particular theory, since type Icell markers were not detected in the majority of the cells culturedwithin lung scaffold in bioreactor, it is possible that additionalstimuli, such as cyclic stretch or exposure to an air-liquid interface,may be necessary to promote expression of more type I alveolar markers(Gutierrez J A, et al. 1998. Am J Physiol 274(2 Pt 1):L196-202,Ostrowski L E, et al. Expt Lung Res 21 (6) 957-970, Alcorn D, et al. JAnat 123(Pt 3):649-660). Collectively, the data presented hereindemonstrate that that in vitro lung regeneration from autologous cellsmay be a viable strategy for tissue repair and cell therapyapplications.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

1-11. (canceled)
 12. A population of lung cells produced by a method ofdifferentiating a stem cell into a lung cell, the method comprising: a)inducing a stem cell into a definitive endoderm cell; b) inducing thedefinitive endoderm cell into an anterior foregut endoderm cell; c)inducing the anterior foregut endoderm cell into a lung cell, therebydifferentiating a stem cell into a lung cell.
 13. The population lungcells of claim 12, wherein the population is at least 95% of cellsexhibiting an alveolar type II phenotype.
 14. The population of lungcells of claim 13, wherein the alveolar type II phenotype is expressionof an alveolar type II cell marker selected from the group consisting ofSPC, Mucin-1, SPB, CD54, and any combination thereof.
 15. The populationof lung cells of claim 12, wherein the population comprises geneticallymodified cells.
 16. The population of lung cells of claim 12, whereinthe cells are genetically modified to express a therapeutic gene. 17.The population of lungs cells of claim 12, wherein the cells resemblefreshly isolated human primary alveolar type II cells.
 18. A method ofalleviating or treating a lung defect in a mammal, the method comprisingadministering to the mammal a therapeutically effective amount of acomposition comprising a population of lung cells produced by a methodof differentiating a stem cell into a lung cell, thereby alleviating ortreating said lung defect in said mammal, wherein the differentiationmethod comprise: a) inducing a stem cell into a definitive endodermcell; b) inducing the definitive endoderm cell into an anterior foregutendoderm cell; c) inducing the anterior foregut endoderm cell into alung cell, thereby differentiating a stem cell into a lung cell.